Dual phase drug release system

ABSTRACT

The present invention relates to conjugate comprising a carrier substituted with one or more occurrences of a moiety having the structure (I): wherein each occurrence of M is independently a modifier having a molecular weight≦10 kDa; denotes direct of indirect attachment of M to linker L M ; and each occurrence of L M  is independently an optionally substituted succinamide-containing linker, whereby the modifier M is directly or indirectly attached to the succinamide linker through an amide bond, and the carrier is linked directly or indirectly to each occurrence of the succinamide linker through an ester bond. In another aspect, the invention provides compositions comprising the conjugates, methods for their preparation, and methods of use thereof in the treatment of various disorder, including, but not limited to cancer.

PRIORITY INFORMATION

The present Application claims priority to U.S. patent application No.60/500,571 filed Sep. 5, 2003, the entire contents of which areincorporated herein by reference.

GOVERNMENT FUNDING

The present invention was made with support, in part, from grants fromthe National Center for Research Resources of the National Institutes ofHealth (Grant Numbers R21-RR14221 and T32 GM07035). Accordingly, theUnited States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Traditionally, pharmaceuticals have primarily consisted of smallmolecules that are dispensed orally (as solid pills and liquids) or asinjectables. Over the past three decades, formulations (i.e.,compositions that control the route and/or rate of drug delivery andallow delivery of the therapeutic agent at the site where it is needed)have become increasingly common and complex. Nevertheless, manyquestions and challenges regarding the development of new treatments aswell as the mechanisms with which to administer them remain to beaddressed.

Although considerable research efforts in this area have led tosignificant advances, drug delivery methods/systems that have beendeveloped over the years and are currently used, still exhibit specificproblems that require improvement. For example, many drugs exhibitlimited or otherwise reduced potencies and therapeutic effects becausethey are either generally subject to partial degradation before theyreach a desired target in the body, or accumulate in tissues other thanthe target, or both.

One objective in the field of drug delivery systems, therefore, is todeliver medications intact to specifically targeted areas of the bodythrough a system that can stabilize the drug and control the in vivotransfer of the therapeutic agent utilizing either physiological orchemical mechanisms, or both. Over the past decade, materials such aspolymeric microspheres, polymer micelles, soluble polymers andhydrogel-type materials have been shown to be effective in enhancingdrug stability in vitro and in vivo, release dynamics, targetingspecificity, lowering systemic drug toxicity, and thus have shown greatpotential for use in biomedical applications, particularly as componentsof various formulations and drug delivery devices.

Therefore a need exists in the biomedical field for low-toxicity,biodegradable, biocompatible, hydrophilic polymer conjugates comprisingpharmaceutically useful modifiers, which overcome or minimize theabove-referenced problems. Such polymer conjugates would find use inseveral applications, including therapeutic and diagnosticpharmaceutical formulations, gene vectors, medical devices, implants,and other therapeutic, diagnostic and prophylatic agents.

The design and engineering of biomedical polymers (e.g., polymers foruse under physiological conditions) are generally subject to specificand stringent requirements. In particular, such polymeric materials mustbe compatible with the biological milieu in which they will be used,which often means that they show certain characteristics ofhydrophilicity. In several applications, they also have to demonstrateadequate biodegradability (i.e., they degrade to low molecular weightspecies. The polymer fragments are in turn metabolized in the body orexcreted,).

Biodegradability is typically accomplished by synthesizing or usingpolymers that have hydrolytically unstable linkages in the backbone. Themost common chemical backbone components with this characteristic areesters and amides. Most recently, novel polymers have been developedwith anhydride, orthoester, polyacetal, polyketal and otherbiodegradable backbone components. Hydrolysis of the backbone structureis the prevailing mechanism for the degradation of such polymers. Otherpolymer types, such as polyethers, may degrade through intra- orextracellular oxidation. Biodegradable polymers can be either natural orsynthetic. Synthetic polymers commonly used in medical applications andbiomedical research include polyethyleneglycol (pharmacokinetics andimmune response modifier), polyvinyl alcohol (drug carrier), andpoly(hydroxypropylmethacrylamide) (drug carrier). In addition, naturalpolymers are also used in biomedical applications. For instance,dextran, hydroxyethylstarch, albumin, polyaminoacids and partiallyhydrolyzed proteins find use in applications ranging from plasmaexpanders, to radiopharmaceuticals to parenteral nutrition. In general,synthetic polymers may offer greater advantages than natural materialsin that they can be tailored to give a wider range of properties andmore predictable lot-to-lot uniformity than can materials from mostnatural sources. Methods of preparing various polymeric materials arewell known in the art. In many biomedical applications, polymermolecules should be chemically associated with the drug substance, orother modifiers, or with each other (e.g., forming a gel). Severalproperties of the final product depend on the character of association,for example, drug release profile, immunotoxicity, immunogenicity andpharmacokinetics. Therefore a need exists for methods of polymerassociation with drug substances and other pharmaceutically usefulmodifiers that would be compatible with the biomedical use of theassociates (conjugates, gels). Such methods should further allow, wherenecessary, drug release in under physiological conditions with anoptimal rate and in a chemical form or forms optimally suited for theintended application.

SUMMARY OF THE INVENTION

The present invention discloses a polymer conjugate that isbiodegradable, biocompatible and exhibits little toxicity and/orbioadhesivity in vivo, and contains one or more modifiers covalentlyattached to the polymer via optionally substitutedsuccinamide-containing linkages.

In one aspect, the invention encompasses a conjugate comprising acarrier substituted with one or more occurrences of a moiety having thestructure:

wherein each occurrence of M is independently a modifier having amolecular weight≦10 kDa;

denotes direct of indirect attachment of M to linker L^(M); and

each occurrence of L^(M) is independently an optionally substitutedsuccinamide-containing linker, whereby the modifier M is directly orindirectly attached to the succinamide linker through an amide bond, andthe carrier is linked directly or indirectly to each occurrence of thesuccinamide linker through an ester bond.

In another aspect, the invention provides compositions comprising theconjugates, methods for their preparation, and methods of use thereof inthe treatment of various disorders, including, but not limited tocancer.

Definitions

Certain compounds of the present invention, and definitions of specificfunctional groups are also described in more detail herein. For purposesof this invention, the chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75^(th) Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,the entire contents of which are incorporated herein by reference.Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups.

“Protecting group”: as used herein, the term protecting group means thata particular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In preferred embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup must be selectively removed in good yield by readily available,preferably nontoxic reagents that do not attack the other functionalgroups; the protecting group forms an easily separable derivative (morepreferably without the generation of new stereogenic centers); and theprotecting group has a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogenand carbon protecting groups may be utilized. For example, in certainembodiments, certain exemplary oxygen protecting groups may be utilized.These oxygen protecting groups include, but are not limited to methylethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM(methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM(p-methoxybenzyloxymethyl ether), to name a few), substituted ethylethers, substituted benzyl ethers, silyl ethers (e.g., TMS(trimethylsilyl ether), TES (triethylsilylether), TIPS(triisopropylsilyl ether), TBDMS (t-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (t-butyldiphenyl silyl ether), to name a few), esters(e.g., formate, acetate, benzoate (Bz), trifluoroacetate,dichloroacetate, to name a few), carbonates, cyclic acetals and ketals.In certain other exemplary embodiments, nitrogen protecting groups areutilized. Nitrogen protecting groups, as well as protection anddeprotection methods are known in the art. Guidance may be found in“Protective Groups in Organic Synthesis” Third Ed. Greene, T. W. andWuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entirecontents of which are hereby incorporated by reference. In certainexemplary embodiments, R^(N1) and R^(N2) are each hydrogen. Nitrogenprotecting groups include, but are not limited to, carbamates (includingmethyl, ethyl and substituted ethyl carbamates (e.g., Troc), to name afew) amides, cyclic imide derivatives, N-Alkyl and N-Aryl amines, iminederivatives, and enamine derivatives, to name a few. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the present invention. Additionally, a variety of protectinggroups are described in “Protective Groups in Organic Synthesis” ThirdEd. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York:1999, the entire contents of which are hereby incorporated by reference.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe compounds that exert minimal destructive or host responseeffects while in contact with body fluids or living cells or tissues.Thus a biocompatible group, as used herein, refers to an aliphatic,alicyclic, heteroaliphatic, heteroalicyclic, aryl or heteroaryl moiety,which falls within the definition of the term biocompatible, as definedabove and herein. The term “Biocompatibility” as used herein, is alsotaken to mean minimal interactions with recognition proteins, e.g.,naturally occurring antibodies, cell proteins, cells and othercomponents of biological systems, unless such interactions arespecifically desirable. Thus, substances and functional groupsspecifically intended to cause the above effects, e.g., drugs andprodrugs, are considered to be biocompatible. Preferably (with exceptionof compounds intended to be cyclotoxic, such as e.g. antineoplasticagents), compounds are “biocompatible” if their addition to normal cellsin vitro, at concentrations similar to the intended systemic in vivoconcentrations, results in less than or equal to 1% cell death duringthe time equivalent to the half-life of the compound in vivo (e.g., theperiod of time required for 50% of the compound administered in vivo tobe eliminated/cleared), and their administration in vivo induces minimaland medically acceptable inflammation, foreign body reaction,immunotoxicity, chemical toxicity or other such adverse effects. In theabove sentence, the term “normal cells” refers to cells that are notintended to be destroyed or otherwise significantly affected by thecompound being tested.

“Biodegradable”: As used herein, “biodegradable” polymers are polymersthat are susceptible to biological processing in vivo. As used herein,“biodegradable” compounds are those that, when taken up by cells, can bebroken down by the lysosomal or other chemical machinery or byhydrolysis into components that the cells can either reuse or dispose ofwithout significant toxic effect on the cells. The degradation fragmentspreferably induce no or little organ or cell overload or pathologicalprocesses caused by such overload or other adverse effects in vivo.Examples of biodegradation processes include enzymatic and non-enzymatichydrolysis, oxidation and reduction. Suitable conditions fornon-enzymatic hydrolysis of the polymer backbones of various conjugates,for example, include exposure of the biodegradable conjugates to waterat a temperature and a pH of lysosomal intracellular compartment.Biodegradation of some conjugate backbones, e.g. polyal conjugates ofthe present invention, can also be enhanced extracellularly, e.g. in lowpH regions of the animal body, e.g. an inflamed area, in the closevicinity of activated macrophages or other cells releasing degradationfacilitating factors. In certain preferred embodiments, the effectivesize of the polymer molecule at pH˜7.5 does not detectably change over 1to 7 days, and remains within 50% of the original polymer size for atleast several weeks. At pH˜5, on the other hand, the polymer preferablydetectably degrades over 1 to 5 days, and is completely transformed intolow molecular weight fragments within a two-week to several-month timeframe. Polymer integrity in such tests can be measured, for example, bysize exclusion HPLC. Although faster degradation may be in some casespreferable, in general it may be more desirable that the polymerdegrades in cells with the rate that does not exceed the rate ofmetabolization or excretion of polymer fragments by the cells. Inpreferred embodiments, the polymers and polymer biodegradationbyproducts are biocompatible.

“Hydrophilic”: The term “hydrophilic” as it relates to substitutents onthe polymer monomeric units does not essentially differ from the commonmeaning of this term in the art, and denotes chemical moieties whichcontain ionizable, polar, or polarizable atoms, or which otherwise maybe solvated by water molecules. Thus a hydrophilic group, as usedherein, refers to an aliphatic, alicyclic, heteroaliphatic,heteroalicyclic, aryl or heteroaryl moiety, which falls within thedefinition of the term hydrophilic, as defined above. Examples ofparticular hydrophilic organic moieties which are suitable include,without limitation, aliphatic or heteroaliphatic groups comprising achain of atoms in a range of between about one and twelve atoms,hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters and polythioesters. In preferredembodiments of the present invention, at least one of the polymermonomeric units include a carboxyl group (COOH), an aldehyde group(CHO), a methylol (CH₂OH) or a glycol (for example, CHOH—CH₂OH orCH—(CH₂OH)₂).

“Hydrophilic”: The term “hydrophilic” as it relates to the polymers ofthe invention generally does not differ from usage of this term in theart, and denotes polymers comprising hydrophilic functional groups asdefined above. In a preferred embodiment, hydrophilic polymer is awater-soluble polymer. Hydrophilicity of the polymer can be directlymeasured through determination of hydration energy, or determinedthrough investigation between two liquid phases, or by chromatography onsolid phases with known hydrophobicity, such as, for example, C4 or C18.

“Biomolecules”: The term “biomolecules”, as used herein, refers tomolecules (e.g., proteins, amino acids, peptides, polynucleotides,nucleotides, carbohydrates, sugars, lipids, nucleoproteins,glycoproteins, lipoproteins, steroids, etc.) which belong to classes ofchemical compounds, whether naturally-occurring or artificially created(e.g., by synthetic or recombinant methods), that are commonly found incells and tissues. Exemplary types of biomolecules include, but are notlimited to, enzymes, receptors, neurotransmitters, hormones, cytokines,cell response modifiers such as growth factors and chemotactic factors,antibodies, vaccines, haptens, toxins, interferons, ribozymes,anti-sense agents, plasmids, DNA, and RNA.

“Carrier”: The term carrier, as used herein, refers to any small orlarge molecule, biomolecule, particle, gel or other object or materialwhich is or can be covalently attached to one or more drug moleculeswith a succinamide linker.

“Physiological conditions”: The phrase “physiological conditions”, asused herein, relates to the range of chemical (e.g., pH, ionic strength)and biochemical (e.g., enzyme concentrations) conditions likely to beencountered in the extracellular fluids of living tissues. For mostnormal tissues, the physiological pH ranges from about 7.0 to 7.4.Circulating blood plasma and normal interstitial liquid representtypical examples of normal physiological conditions.

“Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms“polysaccharide”, “carbohydrate”, or “oligosaccharide” are known in theart and refer, generally, to substances having chemical formula(CH₂O)_(n), where generally n>2, and their derivatives. Carbohydratesare polyhydroxyaldehydes or polyhydroxyketones, or change to suchsubstances on simple chemical transformations, such as hydrolysis,oxydation or reduction. Typically, carbohydrates are present in the formof cyclic acetals or ketals (such as, glucose or fructose). These cyclicunits (monosaccharides) may be connected to each other to form moleculeswith few (oligosaccharides) or several (polysaccharides) monosaccharideunits. Often, carbohydrates with well defined number, types andpositioning of monosaccharide units are called oligosaccharides, whereascarbohydrates consisting of mixtures of molecules of variable numbersand/or positioning of monosaccharide units are called polysaccharides.The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, areused herein interchangeably. A polysaccharide may include natural sugars(e.g., glucose, fructose, galactose, mannose, arabinose, ribose, andxylose) and/or derivatives of naturally occurring sugars (e.g.,2′-fluororibose, 2′-deoxyribose, and hexose).

“Small molecule”: As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively low molecular weight.Preferred small molecules are biologically active in that they produce alocal or systemic effect in animals, preferably mammals, more preferablyhumans. Typically, small molecules have a molecular weight of less thanabout 1500 Da (1500 g/mol). In certain preferred embodiments, the smallmolecule is a drug and the small molecule is referred to as “drugmolecule” or “drug”. In certain embodiment, the drug molecule has MWsmaller or equal to about 10 kDa. Preferably, though not necessarily,the drug is one that has already been deemed safe and effective for useby the appropriate governmental agency or body. For example, drugs forhuman use listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361,and 440 through 460; drugs for veterinary use listed by the FDA under 21C.F.R. §§ 500 through 589, incorporated herein by reference, are allconsidered suitable for use with the present hydrophilic polymers.

Classes of drug molecules that can be used in the practice of thepresent invention include, but are not limited to, anti-cancersubstances, radionuclides, vitamins, anti-AIDS substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents. Many large molecules are also drugs.

A more complete, although not exhaustive, listing of classes andspecific drugs suitable for use in the present invention may be found in“Pharmaceutical Substances: Syntheses, Patents, Applications” by AxelKleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the“Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”,Edited by Susan Budavari et al., CRC Press, 1996, both of which areincorporated herein by reference.

“Pharmaceutically useful group or entity”: As used herein, the termPharmaceutically useful group or entity refers to a compound or fragmentthereof, or a chemical moiety which, when associated with the conjugatesof the present invention, can exert some biological or diagnosticfunction or activity when administered to a subject, or enhance thetherapeutic, diagnostic or preventive properties of the conjugates inbiomedical applications, or improve safety, alter biodegradation orexcretion, or is detectable. Examples of suitable pharmaceuticallyuseful groups or entities include hydrophilicity/hydrophobicitymodifiers, pharmacokinetic modifiers, biologically active modifiers,detectable modifiers.

“Modifier”: As used herein, the term modifier refers to an organic,inorganic or bioorganic moiety that is covalently incorporated into acarrier. Modifiers can be small molecules or macromolecules, and canbelong to any chemical or pharmaceutical class, e.g., nucleotides,chemotherapeutic agents, antibacterial agents, antiviral agents,immunomodulators, hormones or analogs thereof, enzymes, inhibitors,alkaloids and therapeutic radionuclides a therapeutic radionuclide(e.g., alpha, beta or positron emitter). In certain embodiments,chemotherapeutic agents include, but are not limited to, topoisomerase Iand II inhibitors, alkylating agents, anthracyclines, doxorubicin,cisplastin, carboplatin, vincristine, mitromycine, taxol, camptothecin,antisense oligonucleotides, ribozymes, and dactinomycines. In certainembodiments, modifiers according to the invention include, but are notlimited to, biomolecules, small molecules, therapeutic agents,pharmaceutically useful groups or entities, macromolecules, diagnosticlabels, chelating agents, hydrophilic moieties, dispersants, chargemodifying agents, viscosity modifying agents, surfactants, coagulationagents and flocculants, to name a few. A modifier can have one or morepharmaceutical functions, e.g., biological activity and pharmacokineticsmodification. Pharmacokinetics modifiers can include, for example,antibodies, antigens, receptor ligands, hydrophilic, hydrophobic orcharged groups. Biologically active modifiers include, for example,therapeutic drugs and prodrugs, antigens, immunomodulators. Detectablemodifiers include diagnostic labels, such as radioactive, fluorescent,paramagnetic, superparamagnetic, ferromagnetic, X-ray modulating,X-ray-opaque, ultrasound-reflective, and other substances detectable byone of available clinical or laboratory methods, e.g., scintigraphy, NMRspectroscopy, MRI, X-ray tomography, sonotomography, photoimaging,radioimmunoassay. Viral and non-viral gene vectors are considered to bemodifiers.

“Macromolecule”: As used herein, the term macromolecule refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively high molecular weight,generally above 1500 g/mole Preferred macromolecules are biologicallyactive in that they exert a biological function in animals, preferablymammals, more preferably humans. Examples of macromolecules includeproteins, enzymes, growth factors, cytokines, peptides, polypeptides,polylysine, proteins, lipids, polyelectrolytes, immunoglobulins, DNA,RNA, ribozymes, plasmids, and lectins. For the purpose of thisinvention, supramolecular constructs such as viruses, nucleic acidhelices and protein associates (e.g., dimers) are considered to bemacromolecules. When associated with the conjugates of the invention, amacromolecule may be chemically modified prior to being associated withsaid biodegradable biocompatible conjugate.

“Diagnostic label”: As used herein, the term diagnostic label refers toan atom, group of atoms, moiety or functional group, a nanocrystal, orother discrete element of a composition of matter, that can be detectedin vivo or ex vivo using analytical methods known in the art. Whenassociated with a conjugate of the present invention, such diagnosticlabels permit the monitoring of the conjugate in vivo. Alternatively oradditionally, constructs and compositions that include diagnostic labelscan be used to monitor biological functions or structures. Examples ofdiagnostic labels include, without limitation, labels that can be usedin medical diagnostic procedures, such as, radioactive isotopes(radionuclides) for gamma scintigraphy and Positron Emission Tomography(PET), contrast agents for Magnetic Resonance Imaging (MRI) (for exampleparamagnetic atoms and superparamagnetic nanocrystals), contrast agentsfor computed tomography and other X-ray-based imaging methods, agentsfor ultrasound-based diagnostic methods (sonography), agents for neutronactivation (e.g., boron, gadolinium), fluorophores for various opticalprocedures, and, in general moieties which can emit, reflect, absorb,scatter or otherwise affect electromagnetic fields or waves (e.g.gamma-rays, X-rays, radiowaves, microwaves, light), particles (e.g.alpha particles, electrons, positrons, neutrons, protons) or other formsof radiation, e.g. ultrasound.

“Efficient amount of a glycol-specific oxidizing agent”: as it relatesto the oxidative cleavage of the polysaccharides referred to in thepresent invention, the phrase efficient amount of a glycol-specificoxidizing agent means an amount of the glycol-specific oxidizing agentthat provides oxidative opening of essentially all carbohydrate rings ofa polysaccharide.

“Protected hydrophilic group” and “Protected organic moiety” as theseterms are used herein, mean a chemical group which will not interferewith a chemical reaction that the carrier or carrier conjugate issubjected to. Examples of protected hydrophilic groups includecarboxylic esters, alkoxy groups, thioesters, thioethers, vinyl groups,haloalkyl groups, Fmoc-alcohols, etc.

“Aliphatic”: In general, the term aliphatic, as used herein, includesboth saturated and unsaturated, straight chain (i.e., unbranched) orbranched aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. As will be appreciated by one of ordinaryskill in the art, “aliphatic” is intended herein to include, but is notlimited to, alkyl, alkenyl, alkynyl moieties. Thus, as used herein, theterm “alkyl” includes straight and branched alkyl groups. An analogousconvention applies to other generic terms such as “alkenyl”, “alkynyl”and the like. Furthermore, as used herein, the terms “alkyl”, “alkenyl”,“alkynyl” and the like encompass both substituted and unsubstitutedgroups. In certain embodiments, as used herein, “lower alkyl” is used toindicate those alkyl groups (substituted, unsubstituted, branched orunbranched) having about 1-6 carbon atoms.

“Alkenyl”: the term alkenyl denotes a monovalent group derived from ahydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom, which alkenyl groups are optionallysubstituted with one or more functional groups. Substituents include,but are not limited to, any of the substitutents mentioned below, i.e.,the substitutents recited below resulting in the formation of a stablecompound. Alkenyl groups include, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, and the like.

“Alkynyl”: the term alynyl as used herein refers to a monovalent groupderived form a hydrocarbon having at least one carbon-carbon triple bondby the removal of a single hydrogen atom, which alkenyl groups areoptionally substituted. Substituents include, but are not limited to,any of the substitutents mentioned below, i.e., the substitutentsrecited below resulting in the formation of a stable compound.Representative alkynyl groups include ethynyl, 2-propynyl (propargyl),1-propynyl, and the like.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employedin the invention contain about 1-20 aliphatic carbon atoms. In certainother embodiments, the alkyl, alkenyl, and alkynyl groups employed inthe invention contain about 1-10 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain about 1-4 carbon atoms. Illustrative aliphatic groupsthus include, but are not limited to, for example, methyl, ethyl,n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl,n-pentyl, sec-pentyl, isopentyl, tert-pentyl, n-hexyl, sec-hexyl,moieties and the like, which again, may bear one or more substitutents.Alkenyl groups include, but are not limited to, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representativealkynyl groups include, but are not limited to, ethynyl, 2-propynyl(propargyl), 1-propynyl and the like.

“Alicyclic”: as used herein, the term alicyclic refers to compoundswhich combine the properties of aliphatic and cyclic compounds andinclude but are not limited to cyclic, or polycyclic aliphatichydrocarbons and bridged cycloalkyl compounds, which are optionallysubstituted with one or more functional groups. As will be appreciatedby one of ordinary skill in the art, “alicyclic” is intended herein toinclude, but is not limited to, cycloalkyl, cycloalkenyl, andcycloalkynyl moieties, which are optionally substituted with one or morefunctional groups. Illustrative alicyclic groups thus include, but arenot limited to, for example, cyclopropyl, —CH₂-cyclopropyl, cyclobutyl,—CH₂-cyclobutyl, cyclopentyl, —CH₂-cyclopentyl-n, cyclohexyl,—CH₂-cyclohexyl, cyclohexenylethyl, cyclohexanylethyl, norborbylmoieties and the like, which again, may bear one or more substitutents.

“Cycloalkyl”: as used herein, the term cycloalkyl refers specifically togroups having three to seven, preferably three to ten carbon atoms.Suitable cycloalkyls include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, asin the case of aliphatic, heteroaliphatic or heterocyclic moieties, mayoptionally be substituted. An analogous convention applies to othergeneric terms such as “cycloalkenyl”, “cycloalkynyl” and the like.

“Heteroaliphatic”: as used herein, the term heteroaliphatic refers toaliphatic moieties in which one or more carbon atoms in the main chainhave been substituted with a heteroatom. Thus, a heteroaliphatic grouprefers to an aliphatic chain which contains one or more oxygen, sulfur,nitrogen, phosphorus or silicon atoms, e.g., in place of carbon atoms.Heteroaliphatic moieties may be branched or linear unbranched. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; alicyclic; heteroalicyclic; aromatic, heteroaromatic;aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy;heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;heteroarylthio; F; Cl; Br; I; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH;—CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—or -GR^(G1) wherein G is —O—, —S—,—NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2), —OC(═O)—,—NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—,—NR^(G2)C(═O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═NR^(G2))—, —C(═NR^(G2))O—, —C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2)),—NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3)independently includes, but is not limited to, hydrogen, halogen, or anoptionally substituted aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl,or alkylheteroaryl moiety. Additional examples of generally applicablesubstitutents are illustrated by the specific embodiments shown in theExamples that are described herein.

“Heteroalicyclic”, “heterocycloalkyl” or “heterocyclic”: The termheteroalicyclic, heterocycloalkyl or heterocyclic, as used herein,refers to compounds which combine the properties of heteroaliphatic andcyclic compounds and include but are not limited to saturated andunsaturated mono- or polycyclic heterocycles such as morpholino,pyrrolidinyl, furanyl, thiofuranyl, pyrrolyl etc., which are optionallysubstituted with one or more functional groups, as defined herein. Incertain embodiments, the term “heterocyclic” refers to a non-aromatic5-, 6-, 7- or 8-membered ring or a polycyclic group, including, but notlimited to a bi- or tri-cyclic group comprising fused six-membered ringshaving between one and three heteroatoms independently selected fromoxygen, sulfur and nitrogen, wherein (i) each 5-membered ring has 0 to 2double bonds and each 6-membered ring has 0 to 2 double bonds, (ii) thenitrogen and sulfur heteroatoms may optionally be oxidized, (iii) thenitrogen heteroatom may optionally be quaternized, and (iv) any of theabove heterocyclic rings may be fused to an aryl or heteroaryl ring. Incertain embodiments, “heteroalicyclic”, “heterocycloalkyl” or“heterocyclic” refers to a partially unsaturated or fully saturated 3-to 10-membered ring system, which includes single rings of 3 to 8 atomsin size and bi- and tri-cyclic ring systems which may include aromaticsix-membered aryl or aromatic heterocyclic groups fused to anon-aromatic ring. Representative heterocycles include, but are notlimited to, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. Incertain embodiments, a “substituted heterocycloalkyl or heterocycle”group is utilized and as used herein, refers to a heterocycloalkyl orheterocycle group, as defined above, substituted by the independentreplacement of one, two or three of the hydrogen atoms thereon with butare not limited to aliphatic; heteroaliphatic; alicyclic;heteroalicyclic; aromatic, heteroaromatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃;—or -GR^(G1) wherein G is —O—, —S—, —NR^(G2), —C(═O)—,—S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—,—OC(—O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—,—C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) independently includes, but isnot limited to, hydrogen, halogen, or an optionally substitutedaliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic,heteroaromatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety.Additional examples or generally applicable substitutents areillustrated by the specific embodiments shown in the Examples, which aredescribed herein.

Additionally, it will be appreciated that any of the alicyclic orheteroalicyclic moieties described above and herein may comprise an arylor heteroaryl moiety fused thereto. Additional examples of generallyapplicable substitutents are illustrated by the specific embodimentsshown in the Examples that are described herein.

“Aromatic moiety”: as used herein, the term aromatic moiety refers tostable substituted or unsubstituted unsaturated mono- or polycyclichydrocarbon moieties having preferably 3-14 carbon atoms, comprising atleast one ring satisfying the Huckel rule for aromaticity. Examples ofaromatic moieties include, but are not limited to, phenyl, indanyl,indenyl, naphthyl, phenanthryl and anthracyl.

“Heteroaromatic moiety”: as used herein, the term heteroaromatic moietyrefers to stable substituted or unsubstituted unsaturatedmono-heterocyclic or polyheterocyclic moieties having preferably 3-14carbon atoms, comprising at least one ring satisfying the Huckel rulefor aromaticity. Examples of heteroaromatic moieties include, but arenot limited to, pyridyl, quinolinyl, dihydroquinolinyl, isoquinolinyl,quinazolinyl, dihydroquinazolyl, and tetrahydroquinazolyl.

It will also be appreciated that aromatic and heteroaromatic moieties,as defined herein, may be attached via an aliphatic (e.g., alkyl) orheteroaliphatic (e.g., heteroalkyl) moiety and thus also includemoieties such as -(aliphatic)aromatic, -(heteroaliphatic)aromatic,-(aliphatic)heteroaromatic, -(heteroaliphatic)heteroaromatic,-alkyl)aromatic, -(heteroalkyl)aromatic, -(alkyl)heteroaromatic, and-(heteroalkyl)heteroaromatic moieties. Thus, as used herein, the phrases“aromatic or heteroaromatic moieties” and “aromatic, heteroaromatic,-alkyl)aromatic, -(heteroalkyl)aromatic, (heteroalkyl)heteroaromatic,and -(heteroalkyl)heteroaromatic” are interchangeable. Substituentsinclude, but are not limited to, any of the previously mentionedsubstitutents, i.e., the substitutents recited for aliphatic moieties,or for other moieties as disclosed herein, resulting in the formation ofa stable compound.

“Aryl”: as used herein, the term aryl refers to aromatic moieties, asdescribed above, excluding those attached via an aliphatic (e.g., alkyl)or heteroaliphatic (e.g., heteroalkyl) moiety. In certain embodiments ofthe present invention, “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two rings satisfying the Huckel rule foraromaticity, including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl and the like.

“Heteroaryl”: as used herein, the term heteroaryl refers toheteroaromatic moieties, as described above, excluding those attachedvia an aliphatic (e.g., alkyl) or heteroaliphatic (e.g., heteroalkyl)moiety. In certain embodiments of the present invention, the term“heteroaryl”, as used herein, refers to a cyclic unsaturated radicalhaving from about five to about ten ring atoms of which one ring atom isselected from S, O and N; zero, one or two ring atoms are additionalheteroatoms independently selected from S, O and N; and the remainingring atoms are carbon, the radical being joined to the rest of themolecule via any of the ring atoms, such as, for example, pyridyl,pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl,oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl,quinolinyl, isoquinolinyl, and the like.

Substituents for aryl and heteroaryl moieties include, but are notlimited to, any of the previously mentioned substitutents, i.e., thesubstitutents recited for aliphatic moieties, or for other moieties asdisclosed herein, resulting in the formation of a stable compound. Forexample, aryl and heteroaryl groups (including bicyclic aryl groups) canbe unsubstituted or substituted, wherein substitution includesreplacement of one, two or three of the hydrogen atoms thereonindependently with any one or more of the following moieties including,but not limited to: aliphatic; heteroaliphatic; alicyclic;heteroalicyclic; aromatic, heteroaromatic; aryl; heteroaryl; alkylaryl;alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃;—or -GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—,—S(═O)—, —SO₂—, —C(—O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—,—OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—,—C(S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(—NR^(G2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) independently includes, but isnot limited to, hydrogen, halogen, or an optionally substitutedaliphatic, heteroaliphatic, alicyclic, heteroalicyclic, aromatic,heteroaromatic, aryl, heteroaryl, alkylaryl, or alkylheteroaryl moiety.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

“Alkoxy” (or “alkyloxy”): as used herein, the term alkoxy (or alkyloxy)refers to an alkyl group, as previously defined, attached to the parentmolecular moiety through an oxygen atom (“alkoxy”). In certainembodiments, the alkyl group contains about 1-20 aliphatic carbon atoms.In certain other embodiments, the alkyl group contains about 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains about 1-8 aliphatic carbon atoms. In still other embodiments,the alkyl group contains about 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contains about 1-4 aliphatic carbon atoms.Examples of alkoxy groups, include but are not limited to, methoxy,ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentoxy andn-hexoxy.

“Amine”: the term amine refers to a group having the structure —N(R)₂wherein each occurrence of R is independently hydrogen, or an aliphatic,heteroaliphatic, aromatic or heteroaromatic moiety, or the R groups,taken together, may form a heterocyclic moiety. In certain instances, anamine group can be charged (protonized) or quarternized, e.g., —HN⁺(R)₂or —N⁺(R)₃

“Alkylamino”: as used herein, the term alkylamino refers to a grouphaving the structure —NHR′ wherein R′ is alkyl, as defined herein. Theterm “aminoalkyl” refers to a group having the structure NH₂R′—, whereinR′ is alkyl, as defined herein. In certain embodiments, the alkyl groupcontains about 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl group contains about 1-10 aliphatic carbon atoms.In yet other embodiments, the alkyl, alkenyl, and alkynyl groupsemployed in the invention contain about 1-8 aliphatic carbon atoms. Instill other embodiments, the alkyl group contains about 1-6 aliphaticcarbon atoms. In yet other embodiments, the alkyl group contains about1-4 aliphatic carbon atoms. Examples of alkylamino include, but are notlimited to, methylamino, ethylamino, iso-propylamino and the like.

“Carboxylic acid”: The term carboxylic acid as used herein refers to acompound comprising a group of formula —CO₂H.

“Halo, halide and halogen”: The terms halo, halide and halogen as usedherein refer to an atom selected from fluorine, chlorine, bromine, andiodine.

“Methylol”: The term methylol as used herein refers to an alcohol groupof the structure —CH₂OH.

“Hydroxyalkyl”: As used herein, the term hydroxyalkyl refers to an alkylgroup, as defined above, bearing at least one OH group.

“Mercaptoalkyl”: The term mercaptoalkyl as used therein refers to analkyl group, as defined above, bearing at least one SH group

“Acyl”: The term acyl, as used herein, refers to a group comprising acarbonyl group of the formula C═O. Examples of acyl groups include acylhalides, anhydrides, thioesters, amides and carboxylic esters.

“Hydrocarbon”: The term hydrocarbon, as used herein, refers to anychemical group comprising hydrogen and carbon. The hydrocarbon may besubstituted or unsubstituted. The hydrocarbon may be unsaturated,saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.Illustrative hydrocarbons include, for example, methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl,cyclohexyl, methoxy, diethylamino, and the like. As would be known toone skilled in this art, all valencies must be satisfied in making anysubstitutions.

“Substituted”: The terms substituted, whether preceded by the term“optionally” or not, and substitutent, as used herein, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substitutent. When more than one position in any givenstructure may be substituted with more than one substitutent selectedfrom a specified group, the substitutent may be either the same ordifferent at every position. As used herein, the term “substituted” iscontemplated to include all permissible substitutents of organiccompounds. In a broad aspect, the permissible substitutents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substitutents of organiccompounds. Heteroatoms such as nitrogen may have hydrogen substitutentsand/or any permissible substitutents of organic compounds describedherein which satisfy the valencies of the heteroatoms. Examples ofsubstitutents include, but are not limited to aliphatic;heteroaliphatic; alicyclic; heteroalicyclic; aromatic, heteroaromatic;aryl; heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy;heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio;heteroarylthio; F; Cl; Br; I; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH;—CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃;—or -GR^(G1) wherein G is —O—, —S—,—NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—,—NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2), —NR^(G2)C(═O)O—,—NR^(G2)C(═O)NR^(G2), —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═NR^(G2))—, —C(═NR^(G2))O—, —C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—,—NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3)independently includes, but is not limited to, hydrogen, halogen, or anoptionally substituted aliphatic, heteroaliphatic, alicyclic,heteroalicyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl,or alkylheteroaryl moiety. Additional examples of generally applicablesubstitutents are illustrated by the specific embodiments shown in theExamples that are described herein.

“Succinamide linker” or “Succinamide”: unless otherwise specified, asused herein, the term succinamide linker or succinamide designates alinker having the structure:

wherein q is an integer from 0-4; R¹ is hydrogen or a nitrogenprotecting group; and each occurrence of R² is independently hydrogen,halogen, —CN, NO₂, an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl, heteroaryl, aromatic, heteroaromatic moiety, or—GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—,—C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—, —OC(═O)O—,—C(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—, —C(═S)—,—C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) is independently hydrogen,halogen, or an optionally substituted aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aromatic, heteroaromatic, aryl or heteroarylmoiety. In certain embodiments,

designates the site of attachment of a modifier M, which is directly orindirectly attached to the succinamide moiety through an amide bond. Incertain embodiments,

designates the site of attachment of a carrier, which is linked directlyor indirectly to the succinamide moiety through an ester bond.

“Succinimide”: unless otherwise specified, as used herein, the termsuccinimide designates a moiety having the structure:

wherein q is an integer from 0-4; and each occurrence of R² isindependently hydrogen, halogen, —CN, NO₂, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl, heteroaryl, aromatic,heteroaromatic moiety, or -GR^(G1) wherein G is —O—, —S—, —NR^(G2)—,—C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—,—NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—,—NR^(G2)C(═O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═NR^(G2))—, —C(—NR^(G2))O—, —C(NR^(G2))NR^(G3)—, —OC(═NR^(G2))—,—NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3) isindependently hydrogen, halogen, or an optionally substituted aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aromatic, heteroaromatic,aryl or heteroaryl moiety.

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, at any stage of development, including, for example,mammals, birds, reptiles, amphibians, fish, worms and single cells. Cellcultures and live tissue samples are considered to be pluralities ofanimals. Preferably, the non-human animal is a mammal (e.g., a rodent, amouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). Ananimal may be a transgenic animal or a human clone. The term “subject”encompasses animals.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. Preferably, the association is covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, or combinations thereof, etc.

“Efficient amount”: In general, as it refers to an active agent or drugdelivery device, the term “efficient amount” refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the efficient amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, etc. For example, the efficientamount of microparticles containing an antigen to be delivered toimmunize an individual is the amount that results in an immune responsesufficient to prevent infection with an organism having the administeredantigen.

“Directly attached”: as used herein, the term directly attached, as itrefers to covalent attachment of one entity to another (e.g., a modifierattached to a succinamide linker) means that the two entities areconnected via a covalent bond. For example, the present documentdescribes modifiers attached to succinamide linkers, whereby the pointof attachment to the succinamide linker is an amide bond. A suitablemodifier might be any modifier comprising an amine functionality (orprotected form thereof), which forms an amide bond upon reaction withthe carboxylic acid group of a suitable succinic acid linker.

“Indirectly attached”: as used herein, the term indirectly attached, asit refers to covalent attachment of one entity to another (e.g., amodifier attached to a succinamide linker) means that the two entitiesare connected via a linking moiety (as opposed to a direct covalentbond). For example, the present document describes modifiers attached tosuccinamide linkers, whereby the point of attachment to the succinamidelinker is an amide bond. A suitable modifier might be any modifiercomprising a functionality, which may be “capped” with a chemical moietycomprising an amine group; or protected form thereof, such that theamine-capped modifier may now form an amide bond upon reaction with thecarboxylic acid group of a suitable succinic acid linker.

“Natural amino acyl residue”: The term natural amino acyl residue asused herein refers to any one of the common, naturally occurring L-aminoacids found in naturally occurring proteins: glycine (Gly), alanine(Ala), valine (Val), leucine (Leu), isoleucine (Ile), lysine (Lys),arginine (Arg), histidine (His), proline (Pro), serine (Ser), threonine(Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), asparticacid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gln),cysteine (Cys) and methionine (Met).

“Unnatural amino acyl residue”: The term unnatural amino acyl residue asused herein refers to any amino acid which is not a natural amino acid.This includes, for example, α-, β-, ω-, D-, L-amino acyl residues, andcompounds of the general formula

wherein the side chain R is other than the amino acid side chainsoccurring in nature.

“Amino acyl”: More generally, the term amino acyl, as used herein,encompasses natural amino acid and unnatural amino acids.

“PHF” refers to poly(1-hydroxymethylethylene hydroxymethyl-formal).

“CPT” refers to camptothecin.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts an exemplary prodrug release experiment from PHF-CPT inrat plasma at 37° C. Insert: log linearization of PHF-CPT kinetics. Meanvalues from two independent experiments, for all points SD<10% of themean, p<0.05.

FIG. 2 depicts an exemplary tumor size dynamics study in nude mice withLS174t xenografts. Arrows: drug injections (qwx3). Note that even thesmallest conjugate dose is more active than Irinotecan control.Statistics: n=710 per group, standard deviations within 25% of mean; notshown for Figure clarity.

FIG. 3 depicts tumor volume dynamics in surviving animals with LS174txenografts, n=10 per group, equal (160 nm/kg by CPT) doses of Irinotecanand PHF-CPT.

FIG. 4 depicts an exemplary animal survival study corresponding to thetumor size dynamics study of FIG. 3.

FIG. 5 depicts an exemplary biokinetics experiment of PHF-CPT conjugate(¹¹¹In-DTPA labeled PHF backbone and ³H labeled CPT).

FIG. 6 depicts an exemplary biodistribution experiment of the carrierpolymer (¹¹¹In) and CPT (³H) 24 hours post IV administration ofdouble-labeled PHF-CPT. Xenograft: HT29, 0.1-0.15 ml tumors; n=6 pergroup.

FIG. 7 depicts an exemplary microdistribution experiment of CPT in tumortissue 24 hours post administration of PHF-CPT. CPT fluorescence (left)and phase contrast (right) images of the same region. Unstained unfixed15 μm slice. Field: 80×130 μm.

FIG. 8 depicts an exemplary comparative % dose per gram tissuedistribution between CPT and PHF=CPT. HT29 xenograft in nude mice (n=6),administered IV at 20 mg of CPT per kg, 48 hours after injection, % doseper gram tissue. 26× Level Of CPT In Tumor with Fleximer 5× Dose inCirculation with Fleximer-CPT

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

Certain preferred embodiments of the invention will now be moreparticularly described and pointed out in the claims. It will beunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Principlefeatures of the invention may be employed in various embodiments withoutdeparting from the spirit and scope of the invention.

Addressing the need for non-bioadhesive, fully biodegradable solublepolymer conjugates for use in biomedical applications, in one aspect,the present invention provides novel carrier conjugates, whereby thecarrier is chemically modified by covalent attachment of small/large(bio)molecules or other (in)organic moieties (i.e., modifiers) viaoptionally substituted monosuccinamide-containing linkages.

Thus, in certain embodiments, the invention provides a conjugatecomprising a carrier substituted with one or more occurrences of amoiety having the structure:

wherein each occurrence of M is independently a modifier;

denotes direct of indirect attachment of M to linker L^(M); and

each occurrence of L^(M) is independently an optionally substitutedsuccinamide-containing linker, whereby the modifier M is directly orindirectly attached to the succinamide linker through an amide bond, andthe carrier is linked directly or indirectly to each occurrence of thesuccinamide linker through an ester bond.

In certain embodiments, each occurrence of L^(M) independently comprisesa moiety having the structure:

wherein

denotes the site of attachment to the modifier M;

denotes the site of attachment to the carrier; q is an integer from 0-4;R¹ is hydrogen, —C(═O)R^(1A), —C(═O)OR^(1A), —SR^(1A), SO₂R^(1A) or analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,aromatic, heteroaromatic moiety, wherein each occurrence of R^(1A) isindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aromatic, heteroaromatic, aryl orheteroaryl; and each occurrence of R² is independently hydrogen,halogen, —CN, NO₂, an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl, heteroaryl, aromatic, heteroaromatic moiety, or—GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—,—C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—, —OC(═O)O—,—OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—, —C(═S)—,—C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═NR^(G2))NR^(G3)—, —OC(—NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) is independently hydrogen,halogen, or an optionally substituted aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aromatic, heteroaromatic, aryl or heteroarylmoiety.

In certain embodiments, R¹ is hydrogen or alkyl, alkenyl, —C(═O)R^(1A),—C(═O)OR^(1A), —SR^(1A), SO₂R^(1A); wherein each occurrence of R^(1A) isindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aromatic, heteroaromatic, aryl,heteroaryl —C(═O)R^(1B) or —GR^(1G), wherein G is —O—, —S—, —NR^(1G),wherein each occurrence of R^(1B) and R^(1G) is independently hydrogen,or an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic,heteroalicyclic, aryl or heteroaryl moiety. In certain embodiments, R¹is hydrogen.

In certain embodiments, each occurrence of R² is independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic,heteroalicyclic, aromatic, heteroaromatic, aryl, heteroaryl,—C(═O)R^(2A) or —ZR^(2A), wherein Z is —O—, —S—, —NR^(2B), wherein eachoccurrence of R^(2A) and R^(2B) is independently hydrogen, or an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety. In certain embodiments, each occurrence of R² is hydrogen. Incertain embodiments, one or more occurrences of R² is C₁₋₁₀alkyl. Incertain embodiments, one or more occurrences of R² is lower alkyl. Incertain embodiment, one or more occurrences of R² is a hydrophobicgroup. In certain embodiment, one or more occurrences of R² is ahydrophilic group. In certain embodiment, one or more occurrences of R²is an anionic group. In certain embodiment, one or more occurrences ofR¹ is a cationic group. In certain embodiment, one or more occurrencesof R² is a receptor ligand.

In certain embodiments, conjugates of the present invention have thegeneral structure:

wherein R² and q are as defined above,

denotes direct of indirect attachment of M to the succinamide linker;and t is an integer designating the number of modifier moietiesconjugated to the carrier.

Such conjugates feature dual phase release of the modifier moieties (M),as depicted in Scheme 1 below:

The dual phase release proceeds with ester bond cleavage (with releaseof Carrier-OH) and simultaneous M-succinimide formation at the amideside, followed by further hydrolysis of the M-succinimide moiety (withrelease of M). The release process may proceed with formation ofby-products at Phase 1 and/or Phase 2. For example, a by-product thatmay be formed in Phase 1 includes:

where

denotes direct of indirect attachment of M. Similarly, a by-product thatmay be formed in Phase 1 includes:

where

denotes Hydrogen (if direct attachment) or a secondary linker (ifindirect attachment).

In one aspect, the invention encompasses drug-carrier conjugates wherebyone or more drug moiety (e.g., MW smaller than or equal to about 10 kDa)is covalently attached to the carrier via an optionally substitutedsuccinamide linker, either directly or indirectly.

As discussed above, such systems feature dual phase release of the drugmoieties, as depicted in Scheme 2 below:

As discussed above, by-products may be formed in the process.

Attempts have been made to employ succinamidoester linkers with theamide group at the carrier side,⁹ which did not result in dual phasedrug release. In the inventive system, the succinamidoester is orientedsuch that the ester is formed at the carrier side, while the oppositecarboxyl forms an amide bond with an amine-containing modifier (e.g.,drug or drug derivative).

In certain embodiments, the inventive dual phase drug release system, asapplied to drug molecules (i.e., as modifiers) allows the engineering ofsoluble, potentially targetable macromolecular preparations with novelpharmacokinetics and reduced toxicity. In certain embodiments, theinventive system involves assembling of a hydrophilic drug-carrierconjugate that releases a lipophilic prodrug (e.g., CPT prodrug whichhas a stabilized lactone ring), which, in turn, releases the active drugsubstance locally (intra- and extracellularly), without the need forprior metabolization by the hepatic microsomal P450 complex.

Potential advantages of the inventive dual phase release system, asapplied to drugs, include: (1) the ability to prepare water solubledrug-carrier conjugates, which, for example, can be administeredintravenously. (2) activation of the intermediate prodrug (III) “onsite” rather than in the liver, so that local administration andtargeting are possible [Applicant has exemplified this embodiment withCPT as the drug. Unlike other CPT prodrugs, e.g. Irinotecan, theintermediate CPT prodrug is indeed activated “on site”]. (3) theinventive method may allow release of certain drugs in stabilized form(in the form of drug-succinimide intermediate) (as is the case for CPT,which is release in a lactone-stabilized form), which ensures prodrugdeposition in tissues and low rates of redistribution and transfer tourine.

This invention differs from existing drug release systems in at leastthe following ways:

(1) In drug release systems known in the art, the drug is generallyintended to be released in one step. In contrast, the present inventioninvolves (i) release of a succinimidated or drug molecule (prodrug), ora combination of succinimidated and succinamidated forms; and (ii) drugrelease from the succinimidated or succinamidated drug molecule.

(2) In small molecule release systems containing a succinamidate linkergroup between the drug molecule and the carrier known in the art, thedrug is connected with said linker through an ester group. The drug istherefore released in one step, while the linker remains connected tothe carrier.

It should be further understood that consideration should be given tothe size (molecular weight) of Modifiers that may be used in practicingthe present invention. For example, as described in more detail inExample 11, the reaction of cyclization-elimination which results in thesuccinimidated prodrug release involves folding of the succinamidoesterinto a cyclic intermediate structure (See, for example, Schemes 1 and2), with subsequent intramolecular nucleophilic attack on the estercarbon. Without wishing to be bound by any particular theory, sterichindrance from a bulky Modifier (e.g., protein) may prevent such foldingand, therefore, significantly interfere with drug release, making suchmodifiers not suitable for dual phase drug release. In certainembodiments, the present invention describes a class of modifiers thatare suitable for dual phase drug release as modifiers with MW of lessthan 10 kDa, most preferably less than 1.5 kDa. The invention canutilize unsubstituted or substituted succinic acid derivatives, and canbe used in combination with a variety of drug substances, including, butnot limited to, antineoplastic, anti-infective, and anesthetic agents.

Carriers

In certain embodiments, the carrier is any small or large molecule,biomolecule, particle, gel or other object or material which can becovalently attached to one or more modifier (e.g., drug molecule) with amonosuccinamide linker. In certain embodiments, a carrier suitable forpracticing the invention is any small or large molecule, biomolecule,particle, gel or other object or material which comprises one or morefunctionality amenable to succinylation. For example, a suitable carriermight comprise one or more functionality which can react, under suitableconditions, with an optionally substituted succinic anhydride having thestructure:

to form a succinylated carrier having the structure:

or salt thereof;

wherein s is an integer designating the number of succinylation sites onthe carrier; q is an integer from 0-4; and each occurrence of R² isindependently hydrogen, halogen, —CN, NO₂, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl, heteroaryl, aromatic,heteroaromatic moiety, or -GR^(G1) wherein G is —O—, —S—, —NR^(G2)—,—C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—,—NR¹²C(═O)—, —OC(═O)O—, —OC(O)NR^(G2)—, —NR^(G2)C(═O)O—,—NR^(G2)C(O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═NR^(G2))—, —C(—NR^(G2))O—, —C(═O—NR^(G2))NR^(G3)—, —OC(═NR^(G2))—,—NR^(G2)C(—NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3) isindependently hydrogen, halogen, or an optionally substituted aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aromatic, heteroaromatic,aryl or heteroaryl moiety.

In certain other embodiments, a suitable carrier might comprise one ormore functionality which can react, under suitable conditions, with anoptionally substituted succinic anhydride (as above), succinic acid

succinyl dihaloanhydride

succinic ester

or any other reagent suitable for succinylation.

In certain other embodiments, a suitable carrier might comprise one ormore functionality which can react, under suitable conditions, with anoptionally substituted succinic acid having the structure:

wherein q and R² are as defined above;

M is a modifier;

denotes direct of indirect attachment of M to the succinyl moiety; and

R¹ is hydrogen, —C(═O)R^(1A), —C(═O)OR^(1A), —SR^(1A), SO₂R^(1A) or analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,aromatic, heteroaromatic moiety, wherein each occurrence of R^(1A) isindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aromatic, heteroaromatic, aryl orheteroaryl;

to form a conjugate having the structure:

wherein t is an integer designating the number of modifier moietiesconjugated to the carrier.

In certain embodiments, when the carrier is a polymer, about 2 to about25% monomers comprise a modifier M, more preferably about 5 to about20%, more preferably about 5 to about 18%, more preferably about 5 toabout 15%, more preferably about 6 to about 15%, more preferably about 6to about 14%, more preferably about 7 to about 13%, more preferablyabout 7 to about 12%, more preferably about 8 to about 12%, morepreferably about 9 to about 12%, more preferably about 10 to about 12%,more preferably about 9 to about 11%, most preferably about 10 to about11%.

In certain exemplary embodiments, the conjugates of the invention finduse in biomedical applications, such as gene and drug delivery andtissue engineering, and the carrier is biocompatible and biodegradable.In certain embodiments, the carrier is a macromolecule, a molecularmatrix (e.g., a gel or a solid) or an interface. In certain embodiments,the carrier is a macromolecule, soluble polymer, nanoparticle, gel,liposome, micelle, suture, implant, etc. In certain embodiment, the term“soluble polymer” encompasses biodegradable biocompatible polymer suchas a polyal (e.g., hydrophilic polyacetal or polyketal). In certainother embodiments, the carrier is a fully synthetic, semi-synthetic ornaturally-occurring polymer. In certain other embodiments, the carrieris hydrophilic.

In certain exemplary embodiments, the carriers used in the presentinvention are biodegradable biocompatible polyals comprising at leastone hydrolyzable bond in each monomer unit positioned within the mainchain. This ensures that the degradation process (viahydrolysis/cleavage of the monomer units) will result in fragmentationof the polymer conjugate to the monomeric components (i.e.,degradation), and confers to the polymer conjugates of the inventiontheir biodegradable properties. The properties (e.g., solubility,bioadhesivity and hydrophilicity) of biodegradable biocompatible polymerconjugates can be modified by subsequent substitution of additionalhydrophilic or hydrophobic groups. Examples of biodegradablebiocompatible polymers suitable for practicing the invention can befound inter alia in U.S. Pat. Nos. 5,811,510; 5,863,990 and 5,958,398;U.S. Provisional Patent Application 60/348,333; U.S. Utility patentapplication Ser. No. 10/501,565; European Patent Nos.: 0820473 and03707375.6; and International Patent Applications PCT/US03/01017 andPCT/US03/22584, each of the above listed patent documents isincorporated herein by reference in its entirety. Guidance on thesignificance, preparation, and applications of this type of polymers maybe found in the above-cited documents. In certain embodiments, it isanticipated that the present invention will be particularly useful incombination with the above-referenced patent documents, as well as U.S.Pat. No. 5,582,172; U.S. patent application Nos.: 60/147,919 and09/634,320, each of the above listed patent documents is incorporatedherein by reference in its entirety.

As described in the Examples, we have successfully made biodegradablebiocompatible conjugates which are hydrophilic, hydrolyzable andcomprise drug molecules (e.g., camptothecin (i.e., CPT)) covalentlyattached to the polymer carrier via monosuccinamide-containing linkages.Thus, in certain exemplary embodiments, carriers suitable for practicingthe present invention are polyals having at least one acetal/ketaloxygen atom in each monomer unit positioned within the main chain. Asdiscussed above, this ensures that the degradation process (viahydrolysis/cleavage of the polymer acetal/ketal groups) will result infragmentation of the polyal conjugate to low molecular weight components(i.e., degradation). Thus, a novel aspect of the present inventionrelates in part to the structure and properties of conjugates comprisingone or more modifiers covalently attached via succinamide-containinglinkages to a hydrophilic carrier having acetal/ketal groups in the mainchain.

In certain embodiments, biodegradable biocompatible polymer carriers,used for preparation of polymer conjugates of the invention, arenaturally occurring polysaccharides, glycopolysaccharides, and syntheticpolymers of polyglycoside, polyacetal, polyamide, polyether, andpolyester origin and products of their oxidation, fictionalization,modification, cross-linking, and conjugation.

In certain other embodiments, the carrier is a hydrophilic biodegradablepolymer selected from the group consisting of carbohydrates,glycopolysaccharides, glycolipids, glycoconjugates, polyacetals,polyketals, and derivatives thereof.

In certain exemplary embodiments, the carrier is a naturally occurringlinear and branched biodegradable biocompatible homopolysaccharideselected from the group consisting of cellulose, amylose, dextran,levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogen andlixenan.

In certain other exemplary embodiments, the carrier is a naturallyoccurring linear and branched biodegradable biocompatibleheteropolysaccharide selected from the group consisting of agarose,hyluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginicacid and heparin.

In yet other exemplary embodiments, the carrier is a hydrophilic polymerselected from the group consisting of polyacrylates, polyvinyl polymers,polyesters, polyorthoesters, polyamides, polypeptides, and derivativesthereof.

In certain embodiments, the carrier comprises polysaccharides activatedby selective oxidation of cyclic vicinal diols of 1,2-, 1,4-, 1,6-, and2,6-pyranosides, and 1,2-, 1,5-, 1,6-furanosides, or by oxidation oflateral 6-hydroxy and 5,6-diol containing polysaccharides prior toconjugation with one or more modifiers.

In one embodiment, the carriers of the invention comprise activatedhydrophilic biodegradable biocompatible polymer carriers comprising from0.1% to 100% polyacetal moieties represented by the following chemicalstructure:(—O—CH₂—CHR₁—O—CHR₂—)_(n)

wherein R₁ and R₂ are independently hydrogen, hydroxyl, carbonyl,carbonyl-containing substitutent, a biocompatible organic moietycomprising one or more heteroatoms or a protected hydrophilic functionalgroup; and n is an integer from 1-5000.

In still other exemplary embodiments, the carrier comprises abiodegradable biocompatible polyacetal wherein at least a subset of thepolyacetal repeat structural units have the following chemicalstructure:

wherein for each occurrence of the n bracketed structure, one of R¹ andR² is hydrogen, and the other is a biocompatible group and includes acarbon atom covalently attached to C¹; R^(x) includes a carbon atomcovalently attached to C²; n is an integer; each occurrence of R³, R⁴,R⁵ and R⁶ is a biocompatible group and is independently hydrogen or anorganic moiety; and for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a functional groupsuitable for coupling with a succinamide through an ester bond. Incertain embodiments, the functional group is a hydroxyl moiety.

In further exemplary embodiments, the carrier comprises a biodegradablebiocompatible polyketal wherein at least a subset of the polyketalrepeat structural units have the following chemical structure:

wherein each occurrence of R¹ and R² is a biocompatible group andincludes a carbon atom covalently attached to C¹; R^(x) includes acarbon atom covalently attached to C²; n is an integer; each occurrenceof R³, R⁴, R⁵ and R⁶ is a biocompatible group and is independentlyhydrogen or an organic moiety; and for each occurrence of the bracketedstructure n, at least one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises afunctional group suitable for coupling with a succinamide through anester bond. In certain embodiments, the functional group is a hydroxylmoiety.

Examples of suitable organic moieties are aliphatic groups having achain of atoms in a range of between about one and twelve atoms,hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters, polythioesters, pharmaceutically usefulgroups, a biologically active substance or a diagnostic label.

In certain embodiments, in the polyacetals and polyketals describeddirectly above, for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a functional group thatincreases the polymer hydrophilicity or is adapted for covalent bindingto the succinamide linker.

In certain embodiments, in the polyacetals and polyketals describeddirectly above, for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a carbonyl groupadapted for covalent binding to linker L^(M). In certain exemplaryembodiments, the polyacetals and polyketals described directly above,wherein at least one of R¹, R², R³, R⁴, R⁵ and R⁶ comprise a hydroxylgroup, are conjugated with one or more moieties having the structure:

wherein q, and R² are as defined generally above and in classes andsubclasses herein.

In yet another embodiment, at least one of R¹, R², R³, R⁴, R⁵ and R⁶contains a chiral moiety.

In certain embodiments, the biodegradable biocompatible carriers of theinvention can be crosslinked. Guidance for crosslinkers and crosslinkingmethodology in connection with polyals in general may be found, forexample, in U.S. Provisional Application No. 60/348,333; U.S. Utilityapplication Ser. No. 10/501,565 and International Application No.:PCT/US03/01017.

In certain exemplary embodiments, the carrier is a biodegradablebiocompatible polyal that is crosslinked with epibromohydrin, orepichlorohydrin. In certain embodiments, the epibromohydrin orepichlorohydrin is present in an amount in the range of between aboutone and about twenty five percent by weight of the crosslinkedbiodegradable biocompatible polyals.

In one embodiment, biodegradable biocompatible polyals suitable forpracticing the present invention have a molecular weight of betweenabout 0.5 and about 1500 kDa. In a preferred embodiment of the presentinvention, the biodegradable biocompatible polyals have a molecularweight of between about 1 and about 1000 kDa.

In certain embodiments, the polymer carriers are modified (i.e.,conjugated with one or more modifiers) at one or both termini. Forexample, when the carrier is a polyketal, the carrier may have thestructure:

wherein n is an integer and R′, R″ and R′″ may be a modifier. Forexample, R′ can comprise an N-hydroxysuccinimide ester or a maleimidemoiety for conjugation with proteins or other biomolecules; R″ and R′″can comprise a phospholipid and a target specific moiety, such asantibody, respectively, for liposome modification.

In certain other embodiments, carriers can be substituted at oneterminal and one or more non-terminal positions, or at both terminal andone or more non-terminal positions.

In certain embodiments, the carrier is a linear macromolecule, abranched macromolecule, a globular macromolecule, a graft copolymer, acomb copolymer, a nanoparticle or a lipid-based carrier. In certainexemplary embodiments, the lipid-based carrier is a liposome.

In certain embodiments, the carrier is PHF, a structure of which isshown below:

Modifiers

In certain embodiments, modifiers according to the invention include,but are not limited to, biomolecules, small molecules, organic orinorganic molecules, therapeutic agents, microparticles,pharmaceutically useful groups or entities, macromolecules, diagnosticlabels, chelating agents, intercalator, hydrophilic moieties,dispersants, charge modifying agents, viscosity modifying agents,surfactants, coagulation agents and flocculants, to name a few. Incertain embodiment, the modifier is a chemotherapeutic moiety. Incertain embodiments, the modifier is camptothecin (CPT), which isoptionally covalently bound to a secondary linker. In certainembodiments, the modifier is Taxol, which is optionally covalently boundto a secondary linker. In certain embodiments, the modifier is Illudin,which has the structure:

which is optionally covalently bound to a secondary linker.

Examples of biomolecules include, but are not limited to, enzymes,receptors, neurotransmitters, hormones, cytokines, cell responsemodifiers such as growth factors and chemotactic factors, antibodies,vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents,plasmids, DNA, and RNA.

Examples of small molecules include, but are not limited to, drugs suchas vitamins, anti-AIDS substances, ant-cancer substances, radionuclides,antibiotics, immunosuppressants, anti-viral substances, enzymeinhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics and imagingagents.

In certain embodiments, the modifier is a small molecule having amolecular weight preferably ≦about 10 kDa, more preferably ≦about 9 kDa,more preferably ≦about 8 kDa, more preferably ≦about 7 kDa, morepreferably ≦about 6 kDa, more preferably ≦about 5 kDa, more preferably≦about 4 kDa, more preferably ≦about 3 kDa, most preferably ≦about 1.5kDa.

Examples of suitable pharmaceutically useful groups or entities include,but are not limited to, hydrophilicity/hydrophobicity modifiers,pharmacokinetic modifiers, biologically active modifiers and detectablemodifiers.

Examples of diagnostic labels include, but are not limited to,diagnostic radiopharmaceutical or radioactive isotopes for gammascintigraphy and PET, contrast agent for Magnetic Resonance Imaging(MRI) (for example paramagnetic atoms and superparamagneticnanocrystals), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, and moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves, fluorophores in variousoptical procedures, etc. Diagnostic radiopharmaceuticals includeγ-emitting radionuclides, e.g., indium-111, technetium-99m andiodine-131, etc. Contrast agents for MRI (Magnetic Resonance Imaging)include magnetic compounds, e.g. paramagnetic ions, iron, manganese,gadolinium, lanthanides, organic paramagnetic moieties andsuperparamagnetic, ferromagnetic and antiferromagnetic compounds, e.g.,iron oxide colloids, ferrite colloids, etc. Contrast agents for computedtomography and other X-ray based imaging methods include compoundsabsorbing X-rays, e.g., iodine, barium, etc. Contrast agents forultrasound based methods include compounds which can absorb, reflect andscatter ultrasound waves, e.g., emulsions, crystals, gas bubbles, etc.Still other examples include substances useful for neutron activation,such as boron and gadolinium. Further, labels can be employed which canreflect, refract, scatter, or otherwise affect X-rays, ultrasound,radiowaves, microwaves and other rays useful in diagnostic procedures.Fluorescent labels can be used for photoimaging. In certain embodimentsa modifier comprises a paramagnetic ion or group.

In certain embodiments, the modifier may be chemically modified so thatit comprises a functional group (i.e., amine group) suitable forcovalent binding with an optionally substituted succinic acid throughformation of an amide bond; said succinic acid being conjugated to thecarrier through formation of an ester bond.

Conjugates

Conjugates of the invention comprise one or more occurrences of M, whereM is a modifier, wherein the one or more occurrences of M may be thesame or different. In certain embodiments, one or more occurrences of Mis a biocompatible moiety. In certain embodiments, one or moreoccurrences of M is a hydrophilic moiety. In certain embodiments, one ormore occurrences of M is a drug molecule. In certain embodiments, one ormore occurrences of M is a chemotherapeutic moiety. In certainembodiments, one or more occurrences of M is a camptothecin moiety.

In certain other embodiment, one or more occurrences of M is attached tothe succinamide linker either directly or through a secondary linker. Incertain embodiments, the secondary linker is an amino acyl residue, andthe conjugate has the following general structure:

wherein p is an integer from 1-12; t is an integer designating thenumber of modifier moieties conjugated to the carrier; and eachoccurrence of R is independently hydrogen, halogen, —CN, NO₂, analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,aromatic, heteroaromatic moiety, or -GR^(G1) wherein G is —O—, —S—,—NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—,—NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—,—NR^(G2)C(═O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═NR^(G2))—, —C(═NR^(G2))O—, —C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—,—NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3) isindependently hydrogen, halogen, or an optionally substituted aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aromatic, heteroaromatic,aryl or heteroaryl moiety.

In certain embodiments, the secondary linker is an α-amino acyl residue,and the conjugate has the following general structure:

wherein t is an integer designating the number of modifier moietiesconjugated to the carrier; and R designates a natural or unnatural aminoacid side chain.

As discussed more generally above, in certain embodiments, when thecarrier is a polymer, about 2 to about 25% monomers comprise a modifierM, more preferably about 5 to about 20%, more preferably about 5 toabout 18%, more preferably about 5 to about 15%, more preferably about 6to about 15%, more preferably about 6 to about 14%, more preferablyabout 7 to about 13%, more preferably about 7 to about 12%, morepreferably about 8 to about 12%, more preferably about 9 to about 12%,more preferably about 10 to about 12%, more preferably about 9 to about11%, most preferably about 10 to about 11%.

In certain embodiments, M is CPT. In certain embodiments, M is CPT andthe secondary linker is an amino acyl residue. In certain embodiments, Mis CPT and the secondary linker is a glycine residue.

In other embodiments, in the conjugates of the invention, one or moreoccurrences of M comprises a biologically active modifier. In certainexemplary embodiments, one or more occurrence of M is selected from thegroup consisting of proteins, antibodies, antibody fragments, peptides,steroids, intercalators, drugs, hormones, cytokines, enzymes, enzymesubstrates, receptor ligands, lipids, nucleotides, nucleosides, metalcomplexes, cations, anions, amines, heterocycles, heterocyclic amines,aromatic groups, aliphatic groups, intercalators, antibiotics, antigens,immunomodulators, and antiviral compounds. In certain embodiments, thedrugs include, but are not limited to, antineoplastic, antibacterial,antiviral, antifungal, antiparasital, anesthetic drugs.

In certain embodiment, the modifier is a chemotherapeutic moiety. Incertain embodiments, the modifier is camptothecin (CPT). Thus, in oneaspect, the present invention provides a CPT-carrier conjugate, whereinCPT and the carrier are covalently attached through asuccinamide-containing linker, whereby CPT is directly or indirectlyattached to the succinamide moiety through an amide bond, and thecarrier is linked directly or indirectly to the succinamide moietythrough an ester bond. In certain embodiments, CPT is indirectlyattached to the succinamide moiety via an amino acyl moiety. In certainembodiments, CPT is indirectly attached to the succinamide moiety via aglycine moiety. In certain embodiments, the carrier is a polyal, such asthose described herein.

In certain embodiments, there is provided a PHF-CPT conjugate having thestructure (I) shown in Scheme 3 below:

wherein n, k and m are integers between 10-300, 1-20, and 0-300respectively.

As depicted above, conjugate (I) can subsequently release CPT in atwo-phase process.

In certain embodiment, there is provided a PHF-CPT conjugate having thestructure:

wherein n is an integer between 10-3000.

In certain embodiments, there is provided a PHF-Taxol conjugate havingthe structure (II) shown in Scheme 4 below:

wherein n, k and m are integers between 10-300, 1-20, and 0-300respectively.

As depicted above, conjugate (II) can subsequently release Taxol in atwo-phase process.

In certain embodiment, there is provided a PHF-Taxol conjugate havingthe structure:

wherein n is an integer between 10-3000.

In certain embodiments, there is provided a PHF-Illudin conjugate havingthe structure (III) shown in Scheme 5 below:

wherein n, k and m are integers between 10-300, 1-20, and 0-300respectively.

As depicted above, conjugate (III) can subsequently release Illudin in atwo-phase process.

In certain embodiments, there is provided a PHF-Illudin conjugate havingthe structure:

wherein n is an integer between 10-3000.

In certain other embodiments, one or more occurrence of M comprises adetectable label. In certain exemplary embodiments, one or moreoccurrence of M comprises atoms or groups of atoms comprisingradioactive, paramagnetic, superparamagnetic, fluorescent, or lightabsorbing structural domains.

In certain other embodiments, one or more occurrences of M comprise adiagnostic label.

In certain exemplary embodiments, the inventive conjugate comprises abiologically active modifier and a detectable label.

In certain other embodiments, the inventive conjugate comprises adetectable label linked directly to the polymer chain.

The biodegradable biocompatible conjugates of the invention can beprepared to meet desired requirements of biodegradability andhydrophilicity. For example, under physiological conditions, a balancebetween biodegradability and stability can be reached. For instance, itis known that macromolecules with molecular weights beyond a certainthreshold (generally, above 50-100 kDa, depending on the physical shapeof the molecule) are not excreted through kidneys, as small moleculesare, and can be cleared from the body only through uptake by cells anddegradation in intracellular compartments, most notably lysosomes. Thisobservation exemplifies how functionally stable yet biodegradablematerials may be designed by modulating their stability under generalphysiological conditions (pH=7.5±0.5) and at lysosomal pH (pH near 5).For example, hydrolysis of acetal and ketal groups is known to becatalyzed by acids, therefore polyals will be in general less stable inacidic lysosomal environment than, for example, in blood plasma. One candesign a test to compare polymer degradation profile at, for example,pH=5 and pH=7.5 at 37° C. in aqueous media, and thus to determine theexpected balance of polymer stability in normal physiologicalenvironment and in the “digestive” lysosomal compartment after uptake bycells. Polymer integrity in such tests can be measured, for example, bysize exclusion HPLC. One skilled on the art can select other suitablemethods for studying various fragments of the degraded conjugates ofthis invention.

In many cases, it will be preferable that at pH=7.5 the effective sizeof the polymer will not detectably change over 1 to 7 days, and remainwithin 50% from the original for at least several weeks. At pH=5, on theother hand, the polymer should preferably detectably degrade over 1 to 5days, and be completely transformed into low molecular weight fragmentswithin a two-week to several-month time frame. Although fasterdegradation may be in some cases preferable, in general it may be moredesirable that the polymer degrades in cells with the rate that does notexceed the rate of metabolization or excretion of polymer fragments bythe cells. Accordingly, in certain embodiments, the conjugates of thepresent invention are expected to be biodegradable, in particular uponuptake by cells, and relatively “inert” in relation to biologicalsystems. The products of carrier degradation are preferably unchargedand do not significantly shift the pH of the environment. It is proposedthat the abundance of alcohol groups may provide low rate of polymerrecognition by cell receptors, particularly of phagocytes. The polymerbackbones of the present invention generally contain few, if any,antigenic determinants (characteristic, for example, for somepolysaccharides and polypeptides) and generally do not comprise rigidstructures capable of engaging in “key-and-lock” type interactions in,vivo unless the latter are desirable. Thus, the soluble, crosslinked andsolid conjugates of this invention are predicted to have low toxicityand bioadhesivity, which makes them suitable for several biomedicalapplications.

In certain embodiments of the present invention, the biodegradablebiocompatible conjugates can form linear or branched structures. Forexample, the biodegradable biocompatible polyal conjugates of thepresent invention can be chiral (optically active). Optionally, thebiodegradable biocompatible polyal conjugates of the present inventioncan be racemic.

In yet another embodiment, the conjugates of the present invention areassociated with a macromolecule or a nanoparticle. Examples of suitablemacromolecules include, but are not limited to, enzymes, polypeptides,polylysine, proteins, lipids, polyelectrolytes, antibodies, ribonucleicand deoxyribonucleic acids and lectins. The macromolecule may bechemically modified prior to being associated with said biodegradablebiocompatible conjugate. Circular and linear DNA and RNA (e.g.,plasmids) and supramolecular associates thereof, such as viralparticles, for the purpose of this invention are considered to bemacromolecules. In certain embodiments, conjugates of the invention arenon-covalently associated with macromolecules.

In certain embodiments, the conjugates of the invention arewater-soluble. In certain embodiments, the conjugates of the inventionare water-insoluble. In certain embodiments, the inventive conjugate isin a solid form. In certain embodiments, the conjugates of the inventionare colloids. In certain embodiments, the conjugates of the inventionare in particle form. In certain embodiments, the conjugates of theinvention are in gel form. In certain embodiments, the conjugates of theinvention are in a fiber form. In certain embodiments, the conjugates ofthe invention are in a film form.

Applications

In one aspect, an area of application of the present invention is cancertreatment/chemotherapy. However, the scope of application of theinvention is not limited to this area. Other applications will bereadily apparent to the reader.

Despite the significant recent improvements in cancer statistics in theUS, cancer remains one of the major causes of death. The efficacy ofchemotherapy, which is the major therapeutic modality, is still limitedby the toxicity of the available drugs that hinders dose elevation tothe levels resulting in reliable remission. One aspect of the presentinvention relates to the possibility of developing new, considerablymore efficient and less toxic chemotherapeutic preparations. Theinventive system can also be useful in inflammation, pain management,and, generally, in all other areas where various sustained release ortargeting of drugs is beneficial.

Macromolecular drug delivery systems, which have been extensivelystudied over the past two decades, significantly improved thepharmacological properties of several drug substances, and provided newtools for controlling drug delivery to cancer cells.^(18,19) A vastmajority of the antineoplastic drug conjugates reported so far (a) areinactive until the drug substance is released from the macromolecularcarrier, and (b) the drug substance is released, or at least intended tobe released, in one stage.^(20,21) In some cases, the conjugate (e.g.,of a protein) may be active without drug release from the carrier.

Benefits of drug association with carrier macromolecules relate, inpart, to the following factors: (1) solubilization of the drugsubstance; (2) restricted drug substance access to normal interstitiumdue to the large hydrodynamic size of the conjugate, (3) conjugatedelivery to the tumor tissues via the Enhanced Permeability andRetention (EPR) effect,²² and (4) maintenance of sustained drug levelsover periods exceeding cancer cell cycle. In some (more recentlydeveloped) conjugates, the specificity of drug delivery to cancer cellsis further addressed via incorporation of various targeting moieties(e.g., antibodies), and via enzyme-assisted hydrolysis of the linkconnecting the drug molecule to the carrier.^(23,24)

In several preclinical studies, antineoplastic drug conjugates wereshown to be less toxic than respective free drugs.²⁵ Antineoplasticactivity of the conjugates (per unit of the administered drug substance)was usually lower than of unmodified drugs, although in some casessimilar or higher.²⁶ However, conjugates are frequently more effectiveat equitoxic doses, so the partial loss of antineoplastic activity isoutweighed by the lower toxicity and larger maximal tolerated doses.

In one aspect, the dual phase drug release system of the invention addstwo additional major benefits: (1) an added feature of controlledproperties of the released prodrug (e.g., hydrophobicity, affinity tocell components, transmembrane transport, drug activity preservation,redistribution from the release site); and (2) an added possibility toregulate both phases of drug release, (thus, for example, optimizingactive drug levels and release duration vs. cancer cell cycle).

As mentioned above, carriers such as non-bioadhesive, fullybiodegradable soluble polymer conjugates would be highly desirable topractice the present invention.

Synthetic Methods

According to the present invention, any available techniques can be usedto make the inventive conjugates or compositions including them, andintermediates and components (e.g., carriers and modifiers) useful formaking them. For example, semi-synthetic and fully synthetic methodssuch as those discussed in detail below may be used.

Carriers

Methods for preparing polymer carriers (e.g., biocompatible,biodegradable polymer carriers) suitable for conjugation to modifiersare known in the art. For example, synthetic guidance can be found inU.S. Pat. Nos. 5,811,510; 5,863,990 and 5,958,398; U.S. ProvisionalPatent Application 60/348,333; U.S. Utility patent application Ser. No.10/501,565; European Patent Nos.: 0820473 and 03707375.6; andInternational Patent Applications PCT/US03/01017 and PCT/US03/22584. Theskilled practitioner will know how to adapt these methods to makepolymer carriers for use in the practice of the invention.

For example, semi-synthetic polyals may be prepared from polyaldoses andpolyketoses via complete lateral cleavage of carbohydrate rings withperiodate in aqueous solutions, with subsequent conversion intohydrophilic moieties (e.g. via borohydride reduction) for conjugation ofhydroxyl groups with one or more modifiers, via a succinamide linker. Inan exemplary embodiment, the carbohydrate rings of a suitablepolysaccharide can be oxidized by glycol-specific reagents, resulting inthe cleavage of carbon-carbon bonds between carbon atoms that are eachconnected to a hydroxyl group. An example of application of thismethodology to dextran B-512 is illustrated below:

A similar approach may be used with Levan:

and Inulin:

In one embodiment, a method for forming the biodegradable biocompatiblepolyal conjugates of the present invention comprises a process by whicha suitable polysaccharide is combined with an efficient amount of aglycol-specific oxidizing agent to form an aldehyde intermediate. Thealdehyde intermediate, which is a polyal itself, may then be reduced tothe corresponding polyol, succinulated, and coupled with one or moresuitable modifiers to form a biodegradable biocompatible polyalconjugate comprising succinamide-containing linkages.

In another preferred embodiment, fully synthetic biodegradablebiocompatible polyals for used in the present invention can be preparedby reacting a suitable initiator with a suitable precursor compound.

For example, fully synthetic polyals may be prepared by condensation ofvinyl ethers with protected substituted diols. Other methods, such ascycle opening polymerization, may be used, in which the method efficacymay depend on the degree of substitution and bulkiness of the protectivegroups.

One of ordinary skill in the art will appreciate that solvent systems,catalysts and other factors may be optimized to obtain high molecularweight products.

In certain embodiments, the carrier is PHF having the structure:

Modifiers

In certain embodiments, modifiers according to the invention include,but are not limited to, biomolecules, small molecules, organic orinorganic molecules, therapeutic agents, microparticles,pharmaceutically useful groups or entities, macromolecules, diagnosticlabels, chelating agents, intercalator, hydrophilic moieties,dispersants, charge modifying agents, viscosity modifying agents,surfactants, coagulation agents and flocculants, to name a few.

As discussed above, modifiers useful in the practice of the inventionmay be chemically modified so that they independently comprise afunctional group suitable for covalent binding with an optionallysubstituted succinic acid through formation of an amide bond; saidsuccinic acid being conjugated to the carrier through formation of anester bond.

Conjugates

In another aspect, the invention provides a method for preparing aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

wherein each occurrence of M is independently a modifier;

denotes direct of indirect attachment of M to linker L^(M); and

each occurrence of L^(M) is independently an optionally substitutedsuccinamide-containing linker, whereby the modifier M is directly orindirectly attached to the succinamide linker through an amide bond, andthe carrier is linked directly or indirectly to each occurrence of thesuccinamide linker through an ester bond;

said method comprising steps of:

providing a carrier;

providing one or more modifiers;

reacting the carrier with an optionally substituted succinic anhydridehaving the structure:

wherein q is an integer from 0-4; and each occurrence of R² isindependently hydrogen, halogen, —CN, NO₂, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl, heteroaryl, aromatic,heteroaromatic moiety, or -GR^(G1) wherein G is —O—, —S—, —NR^(G2)—,—C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—,—NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—,—NR^(G2)C(═O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(—S)S—,—C(═NR^(G2))—, —C(═NR^(G2))O—, —C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—,—NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3) isindependently hydrogen, halogen, or an optionally substituted aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aromatic, heteroaromatic,aryl or heteroaryl moiety;

under suitable conditions to form a succinylated carrier having thestructure:

or salt thereof;

wherein s denotes the number of succinyl moieties on the carrier; and

reacting the succinylated carrier with one or more modifier moieties (M,whereby at least one modifier moiety forms an amide bond, eitherdirectly or indirectly through a secondary linker, with a succinylmoiety present on the carrier; thereby generating the conjugate havingthe structure:

wherein R² and q are as defined above;

denotes direct of indirect attachment of M to the succinamide linker;and t is an integer designating the number of modifier moietiesconjugated to the carrier such that t≦s.

In certain embodiments, each occurrence of R² is independently hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, heterocycloalkynyl, heteroaliphatic,heteroalicyclic, aromatic, heteroaromatic, aryl, heteroaryl,—C(═O)R^(2A) or -ZR^(2A), wherein Z is —O—, —S—, —NR^(2B), wherein eachoccurrence of R^(2A) and R^(2B) is independently hydrogen, or an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety. In certain embodiments, each occurrence of R² is hydrogen. Incertain embodiment, one or more occurrences of R² is a C₁₋₁₀ alkylmoiety. In certain embodiment, one or more occurrences of R² is loweralkyl. In certain embodiment, one or more occurrences of R² is ahydrophobic group. In certain embodiment, one or more occurrences of R²is a hydrophilic group. In certain embodiment, one or more occurrencesof R² is an anionic group. In certain embodiment, one or moreoccurrences of R² is a cationic group. In certain embodiment, one ormore occurrences of R² is a receptor ligand.

In certain exemplary embodiments, in the step of coupling thesuccinylated carrier, a subset of the succinamic acid sites on thecarrier remains unreacted.

In certain embodiments, the degree of succinylation on the carrier ismodulated by varying the ratio of succinic anhydride amount vs. carrieramount in the step of reacting the carrier with the optionallysubstituted succinic anhydride. Thus, succinylation may be controlled byselecting the appropriate succinic anhydride/carrier ratio.

In certain embodiments, the degree of modifier incorporation in theconjugate is modulated by varying the ratio of modifier amount vs.succinylated carrier amount in the step of reacting the succinylatedcarrier with one or more modifier moieties. Thus, modifier contents inthe conjugate may be controlled by selecting the appropriatemodifier/succinylated carrier ratio. In certain embodiments, the degreeof modifier incorporation in the conjugate is determined by the degreeof carrier succinylation.

In certain exemplary embodiments, in practicing the method of theinvention, the carrier is a biodegradable biocompatible polyal such asthose disclosed in U.S. Pat. Nos. 5,811,510; 5,863,990 and 5,958,398;U.S. Provisional Patent Application 60/348,333; U.S. Utility patentapplication Ser. No. 10/501,565; European Patent Nos.: 0820473 and03707375.6; and International Patent Applications PCT/US03/01017 andPCT/US03/22584.

In certain embodiments, a variety of modifiers can be mixed togetherwith the succinylated carrier and the reaction mixture incubated insuitable conditions until the desirable conversion degree is achieved.This method can be used, via mixing the modifiers and the carrier atdifferent ratios, to produce, in one step, libraries of conjugates withvarying modifier composition and content.

In certain embodiment, the modifier is a chemotherapeutic moiety. Incertain embodiments, the modifier is camptothecin (CPT), Taxol orIlludin. Thus, in one aspect, the present invention provides a methodfor preparing a CPT-, Taxol- or Illudin-carrier conjugate, wherein CPT,Taxol or Illudin and the carrier are covalently attached through asuccinamide-containing linker, whereby CPT, Taxol or Illudin is directlyor indirectly attached to the succinamide moiety through an amide bond,and the carrier is linked directly or indirectly to the succinamidemoiety through an ester bond. In certain embodiments, CPT, Taxol orIlludin is indirectly attached to the succinamide moiety via an aminoacyl moiety. In certain embodiments, CPT, Taxol or Illudin is indirectlyattached to the succinamide moiety via a glycine moiety. In certainembodiments, the carrier is a polyal, such as those described herein. Incertain exemplary embodiments, the polyal is PHF. In certainembodiments, a PHF-CPT conjugate according to the present invention canbe prepared as follows:

In certain embodiments, a PHF-Taxol conjugate according to the presentinvention can be prepared as follows:

In certain embodiments, a PHF-Illudin conjugate according to the presentinvention can be prepared as follows:

Compositions

In certain embodiments, there is provided a composition comprising anyone or more of the conjugates disclosed herein and a pharmaceuticallysuitable carrier or diluent. In certain embodiments, the compositioncomprises a CPT-carrier conjugate. In certain embodiments, CPT isindirectly attached to the succinamide moiety via an amino acyl moiety.In certain embodiments, CPT is indirectly attached to the succinamidemoiety via a glycine moiety. In certain embodiments, the carrier is apolyal, such as those described herein. In certain exemplaryembodiments, the polyal is PHF. In certain embodiments, the compositioncomprises a PHF-CPT conjugate having formula (I) or (Ia).

In certain embodiments, the composition comprises a Taxol-carrierconjugate. In certain embodiments, Taxol is indirectly attached to thesuccinamide moiety via an amino acyl moiety. In certain embodiments,Taxol is indirectly attached to the succinamide moiety via a glycinemoiety. In certain embodiments, the carrier is a polyal, such as thosedescribed herein. In certain exemplary embodiments, the polyal is PHF.In certain embodiments, the composition comprises a PHF-Taxol conjugatehaving formula (II) or (IIa).

In certain embodiments, the composition comprises a Illudin-carrierconjugate. In certain embodiments, Illudin is indirectly attached to thesuccinamide moiety via an amino acyl moiety. In certain embodiments,Illudin is indirectly attached to the succinamide moiety via a glycinemoiety. In certain embodiments, the carrier is a polyal, such as thosedescribed herein. In certain exemplary embodiments, the polyal is PHF.In certain embodiments, the composition comprises a PHF-Illudinconjugate having formula (III) or (IIIa).

In certain embodiments, the invention provides a composition in the formof a gel of the inventive biodegradable biocompatible conjugate and abiologically active compound disposed within the gel. Alternatively oradditionally, a diagnostic label can be disposed within the gel or boundto the gel matrix.

In another embodiment, the invention provides a composition in the formof a solution of the biodegradable biocompatible polyal conjugate and apharmaceutically useful entity, a drug or a macromolecule dissolvedwithin the solution. Alternatively or additionally, a diagnostic labelcan be dissolved within the solution.

In certain embodiments, there is provided a composition comprising abiodegradable biocompatible conjugate of the invention associated withan efficient amount of a therapeutic agent; wherein the therapeuticagent is incorporated into and released from said biodegradablebiocompatible conjugate matrix by degradation of the polymer matrix ordiffusion of the agent out of the matrix over a period of time. Incertain embodiments, the conjugate is non-covalently associated with anefficient amount of a therapeutic agent. In certain embodiments, thetherapeutic agent is selected from the group consisting of vitamins,anti-AIDS substances, anti-cancer substances, radionuclides,antibiotics, immunosuppressants, anti-viral substances, enzymeinhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents, and combination thereof.

In variations of these embodiments, it may be desirable to include otherpharmaceutically active compounds, such as antiinflammatories orsteroids which are used to reduce swelling, antibiotics, antivirals, orantibodies. Other compounds which can be included are preservatives,antioxidants, and fillers, coatings or bulking agents which may also beutilized to alter polymer matrix stability and/or drug release rates.

Additives Used to Alter Properties of Conjugate Compositions:

In a preferred embodiment, only conjugate is incorporated into thedelivery device or construct, although other biocompatible, preferablybiodegradable or metabolizable, materials can be included forprocessing, preservation and other purposes, such as buffers andfillers.

Buffers, acids and bases are used to adjust the pH of the composition.Agents to increase the diffusion distance of agents released from theimplanted polymer can also be included.

Fillers are water soluble or insoluble materials incorporated into theformulation to add bulk. Types of fillers include, but are not limitedto, NaCl, mannitol, sugars, synthetic polymers, modified starches andcelluloses. The amount of filler in the formulation will typically be inthe range of between about 1 and about 90% by weight.

Methods of Use

The present invention encompasses polymer conjugates for use inbiomedical applications, primarily (but not exclusively) in the fieldsof pharmacology, bioengineering, wound healing, anddermatology/cosmetics. In certain embodiments, the polymer conjugatesare biodegradable polyal conjugates. In particular, medical applicationsfor the conjugates of the invention include injectable therapeuticpharmaceuticals, injectable diagnostic pharmaceuticals, gels, surgicalimplants, systems for controlled drug release, wound closureapplications (sutures, staples), orthopedic fixation devices (pins,rods, screws, tacks, ligaments), cardiovascular applications (stents,grafts), and as long circulating and targeted drugs. Conjugates of thepresent invention can be employed as components of biomaterials, drugs,drug carriers, pharmaceutical formulations, medical devices, implants,and can be associated with small molecules, pharmaceutically usefulentities, drugs, macromolecules and diagnostic labels.

Methods of Treating

In certain preferred embodiments of the invention, the conjugates of theinvention are used in methods of treating animals (preferably mammals,most preferably humans). In one embodiment, the conjugates of thepresent invention may be used in a method of treating animals whichcomprises administering to the animal a biodegradable biocompatibleconjugate of the invention. For example, conjugates in accordance withthe invention can be administered in the form of soluble linearpolymers, copolymers, conjugates, colloids, particles, gels, soliditems, fibers, films, etc. Biodegradable biocompatible conjugates ofthis invention can be used as drug carriers and drug carrier components,in systems of controlled drug release, preparations for low-invasivesurgical procedures, etc. Pharmaceutical formulations can be injectable,implantable, etc.

In yet another aspect, the invention provides a method of treating adisease or disorder in a subject in need thereof, comprisingadministering to the subject an efficient amount of at least oneconjugate of the invention; wherein said conjugate releases one or moremodifiers in a dual phase process; wherein said modifier(s) is(are)suitable therapeutic agent(s) for the treatment of the disease ordisorder.

In yet another aspect, the invention provides a method of treating adisease or disorder in a subject in need thereof, comprisingadministering to the subject an efficient amount of at least oneconjugate of the invention; wherein said conjugate is associated with atherapeutic agent; whereby:

the conjugate releases one or more modifiers in a dual phase process;wherein said modifier(s) is(are) suitable therapeutic agent(s) for thetreatment of the disease or disorder; and

wherein the therapeutic agent is incorporated into and released frombiodegradable biocompatible polyketal matrix by degradation of thepolymer matrix or diffusion of the agent out of the matrix over a periodof time.

In certain embodiments, the modifier is locally delivered byimplantation of said conjugate matrix at the desired site of delivery.

In certain other exemplary embodiments, any or more of the methodsdescribed above further comprises administering at least one additionalbiologically active compound.

In certain embodiments, the modifier, biologically active compound andtherapeutic agent are independently selected from the group consistingof vitamins, anti-AIDS substances, anti-cancer substances,radionuclides, antibiotics, immunosuppressants, anti-viral substances,enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents, and combination thereof.

In certain embodiments, in practicing the method of the invention, theconjugate further comprises or is associated with a diagnostic label. Incertain exemplary embodiments, the diagnostic label is selected from thegroup consisting of: radiopharmaceutical or radioactive isotopes forgamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves and fluorophores. Incertain exemplary embodiments, the conjugate is further monitored invivo.

In another aspect, the invention provides a method of treating a diseaseor disorder in a subject, comprising preparing an aqueous formulation ofat least one conjugate of the invention and parenterally injecting saidformulation in the subject. In certain exemplary embodiments, theconjugate comprises a biologically active modifier. In certain exemplaryembodiments, the conjugate comprises a detectable modifier.

In another aspect, the invention provides a method of treating a diseaseor disorder in a subject, comprising preparating an implant comprisingat least one conjugate of the invention, and implanting said implantinto the subject. In certain exemplary embodiments, the implant is abiodegradable gel matrix.

In another aspect, the invention provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above, wherein said conjugate comprises abiologically active modifier. In certain exemplary embodiments, thebiologically active component is a gene vector.

In another aspect, the invention provides a method for eliciting animmune response in an animal, comprising administering a conjugate as inthe methods described above, wherein said conjugate comprises an antigenmodifier.

In another aspect, the invention provides a method of diagnosing adisease in an animal, comprising steps of:

administering a conjugate as in the methods described above, whereinsaid conjugate comprises a detectable modifier; and

detecting the detectable modifier.

In certain exemplary embodiments, the step of detecting the detectablemodifier is performed non-invasively. In certain exemplary embodiments,the step of detecting the detectable modifier is performed usingsuitable imaging equipment.

In one embodiment, a method for treating an animal comprisesadministering to the animal the biodegradable biocompatible conjugatesof the invention as a packing for a surgical wound from which a tumor orgrowth has been removed. The biodegradable biocompatible conjugatepacking will replace the tumor site during recovery and degrade anddissipate as the wound heals.

In certain embodiments, the conjugate is associated with a diagnosticlabel for in vivo monitoring.

The conjugates described above can be used for therapeutic,preventative, and analytical (diagnostic) treatment of animals. Theconjugates are intended, generally, for parenteral administration, butin some cases may be administered by other routes.

In one embodiment, soluble or colloidal conjugates are administeredintravenously. In another embodiment, soluble or colloidal conjugatesare administered via local (e.g., subcutaneous, intramuscular)injection. In another embodiment, solid conjugates (e.g., particles,implants, drug delivery systems) are administered via implantation orinjection.

In one embodiment, conjugates comprising a biologically active substance(e.g., a drug or a gene vector) are administered to treat diseaseresponsive to said substance.

In another embodiment, conjugates comprising a detectable label areadministered to study the patterns and dynamics of label distribution inanimal body.

In another embodiment, conjugates comprising an antigen or anantigen-generating component (e.g., a plasmid) are administered todevelop immunity to said antigen.

In certain embodiments, any one or more of the conjugates disclosedherein may be used in practicing any of the methods described above. Incertain exemplary embodiments, the conjugate is a CPT-, Taxol- orIlludin-PHF conjugate.

Applications to Drug Delivery Methods

Examples of applications to drug delivery methods applicable to thepresent invention can be found inter alia in U.S. Provisional PatentApplication 60/348,333; U.S. Utility patent application Ser. No.10/501,565; European Patent No.: 03707375.6; and International PatentApplications PCT/US03/01017. These include Polyal-small-molecule-drugconjugates, protein-modified carriers, Cationized polyal,Polyal-modified liposomes, Polyal-modified nano- and microparticles.

In another embodiment, the biodegradable biocompatible conjugates of thepresent invention can be monitored in vivo by suitable diagnosticprocedures. Such diagnostic procedures include nuclear magneticresonance imaging (NMR), magnetic resonance imaging (MRI), ultrasound,X-ray, scintigraphy, positron emission tomography (PET), etc. Thediagnostic procedure can detect, for example, conjugate disposition(e.g., distribution, localization, density, etc.) or the release ofdrugs, prodrugs, biologically active compounds or diagnostic labels fromthe biodegradable biocompatible conjugate over a period of time.Suitability of the method largely depends on the form of theadministered conjugate and the presence of detectable labels. Forexample, the size and shape of conjugate implants can be determinednon-invasively by NMR imaging, ultrasound tomography, or X-ray(“computed”) tomography. Distribution of soluble conjugate preparationcomprising a gamma emitting or positron emitting radiotracer can beperformed using gamma scintigraphy or PET, respectively.Microdistribution of conjugate preparation comprising a fluorescentlabel can be investigated using photoimaging.

It is understood, for the purpose of this invention, that transfer anddisposition of conjugates in vivo can be regulated by modifying groupsincorporated into the conjugate structure, such as hydrophobic andhydrophilic modifiers, charge modifiers, receptor ligands, antibodies,etc. Such modification, in combination with incorporation of diagnosticlabels, can be used for development of new useful diagnostic agents. Thelatter can be designed on a rational basis (e.g., conjugates of large orsmall molecules binding known tissue components, such as cell receptors,surface antigens, etc.), as well as through screening of libraries ofconjugate molecules modified with a variety of moieties with unknown orpoorly known binding activities, such as synthetic peptides andoligonucleotides, small organic and metalloorganic molecules, etc.

Interface Component

In one embodiment of the present invention, the biodegradablebiocompatible conjugate can be used as an interface component. The term“interface component” as used herein, means a component, such as acoating or a layer on an object, that alters the character of theobject's interaction with the biological milieu, for example, tosuppress foreign body reactions, decrease inflammatory response,suppress clot formation, etc. It should be understood that the objectcan be microscopic or macroscopic. Examples of microscopic objectsinclude macromolecules, colloids, vesicles, liposomes, emulsions, gasbubbles, nanocrystals, etc. Examples of macroscopic objects includesurfaces, such as surfaces of surgical equipment, test tubes, perfusiontubes, items contacting biological tissues, etc. It is believed thatinterface components can, for example, provide the object protectionfrom direct interactions with cells and opsonins and, thus, to decreasethe interactions of the object with the biological system.

Surfaces can be modified by the biodegradable biocompatible conjugate ofthe present invention by, for example, conjugating functional groups ofthe conjugate polymer backbone with functional groups present on thesurface to be modified. For example, aldehyde groups of biodegradablebiocompatible polyal precursors can be reacted with amino groups presenton the surface in the presence of cyanoborohydride to form aminelinkages. In another embodiment, a biodegradable biocompatible polyalconjugate of the invention which includes a suitable terminal group canbe synthesized, such as a polyal having a terminal aldehyde group. Apolymer can be connected to a surface by reaction of the terminal group.

In still another embodiment, a suitable polysaccharide can be linkedwith a surface by reaction of a reducing or non-reducing end of thepolysaccharide or otherwise, by subsequent oxidation/reduction sequenceand further conversion of the remainder of the polysaccharide to producea polyal conjugate.

It is to be understood that the biodegradable biocompatible conjugatesof this invention can be conjugated with macromolecules, such asenzymes, polypeptides, proteins, etc., by the methods described abovefor conjugating the biodegradable biocompatible conjugates withfunctional groups present on a surface.

The biodegradable biocompatible conjugates of the invention can also beconjugated with a compound that can physically attach to a surface via,for example, hydrophobic, van der Waals, and electrostatic interactions.For example, the biodegradable biocompatible polyal precursors can beconjugated with lipids, polyelectrolytes, proteins, antibodies, lectins,etc.

In other embodiments of the present invention, biomedical preparationsof the biodegradable biocompatible polyal conjugates of the inventioncan be made in various forms. It is believed that interface componentscan prolong circulation of macromolecular and colloidal drug carriers.Therefore, small molecules, biologically active compounds, diagnosticlabels, etc., being incorporated in such carriers, can circulatethroughout the body without stimulating an immunogenic response andwithout significant interactions with cell receptors and recognitionproteins (opsonins). Accordingly, a conjugate of this invention can befurther modified with an interface component (e.g., a polymer, such aspolyethyleneglycol or a hydrophilic polyal) such that the drug carryingbackbone of the conjugate is surrounded by a “brush” formed by thechains of said interface component. The latter can be additionallymodified to enable conjugate targeting to a certain molecularmarker,cell or tissue in vivo.

Throughout this document, various publications are referred to, each ofwhich is hereby incorporated by reference in its entirety in an effortto more fully describe the state of the art to which the inventionpertains.

The invention will now be further and specifically described by thefollowing examples. All parts and percentages are by weight unlessotherwise stated.

EQUIVALENTS

The representative examples that follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art.

The following examples contain important additional information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and the equivalents thereof.

EXEMPLIFICATION

The practitioner has a well-established literature of polymer chemistryto draw upon, in combination with the information contained herein, forguidance on synthetic strategies, protecting groups, and other materialsand methods useful for the synthesis of the conjugates of thisinvention.

The various references cited herein provide helpful backgroundinformation on preparing polymers similar to the inventive compoundsdescribed herein or relevant intermediates, as well as information onformulation, uses, and administration of the conjugates of theinvention, which may be of interest. Additional guidance may be foundinter alia in (i) Papisov M I. Acyclic polyacetals from polysaccharides.ACS Symposium Series 786 (2001), 301-314; (ii) Cabodi S., Nenci A., OngL, Papisov M, Dotto G-P. Targeted drug delivery to breast cancer cells.Proceedings, Dept of Defense Breast Cancer Research Program Meeting,Atlanta, Ga., 2000; v. 1 p. 307; (iii) M. I. Papisov, M. Yin, A.Yurkovetskiy, A. Hiller, S. Choi, A. J. Fischman. Fully biodegradablehydrophilic polyals (polyacetals and polyketals). 29th Int. Symp. onControlled Release of Bioactive Materials, 2002, Seoul, Korea.Controlled Release Society, Deerfield, Ill., 2002; paper # 465; (iv) A.Yurkovetskiy, S. Choi, A. Hiller, M. Yin, A. J. Fischman, M. I. Papisov.Biodegradable polyal carriers for protein modification. 29th Int. Symp.on Controlled Release of Bioactive Materials, 2002, Seoul, Korea.Controlled Release Society, Deerfield, Ill., 2002; paper # 357; (v) M.Papisov, A. Yurkovetskiy, M. Yin, A. Hiller, A. J. Fischman. Fullybiodegradable hydrophilic polyacetals for macromolecularradiopharmaceuticals. 49-th Annual Meeting of The Society of NuclearMedicine, Los Angeles, Calif., 2002. J. Nuc. Med. 2002, 43:5(Supplement) p. 377P; (vi) A. V. Yurkovetskiy, A. Hiller, M. Yin, S.Sayed, A. J. Fischman, M. I. Papisov. Biodegradable polyals for proteinmodification. Controlled Release Society's Winter Symposium, Salt LakeCity, Utah, 2003; (vii) Papisov, A Yurkovetskiy, M Yin, P Leone, Alan J.Fischman, Alexander Hiller, and Sakina Sayed. Hydrophilic Polyals:Biomimetic Biodegradable Stealth Materials for Pharmacology andBioengineering. Proceedings of 226th Natl. Meeting of American ChemicalSociety, New York, N.Y., 2003; (viii) A. V. Yurkovetskiy, A. Hiller, S.Syed, M. Yin, X. M. Lu, A. J. Fischman, and M. I. Papisov. Synthesis ofa macromolecular camptothecin conjugate with dual phase drug release.Molecular Pharmacology, 2004, in print.

Moreover, the practitioner is directed to the specific guidance andexamples provided in this document relating to various exemplaryconjugates and intermediates thereof.

The conjugates of this invention and their preparation can be understoodfurther by the examples that illustrate some of the processes by whichthese compounds are prepared or used. It will be appreciated, however,that these examples do not limit the invention. Variations of theinvention, now known or further developed, are considered to fall withinthe scope of the present invention as described herein and ashereinafter claimed.

According to the present invention, any available techniques can be usedto make or prepare the inventive conjugates or compositions includingthem. For example, a variety of solution phase synthetic methods such asthose discussed in detail below may be used. Alternatively oradditionally, the inventive conjugates may be prepared using any of avariety combinatorial techniques, parallel synthesis and/or solid phasesynthetic methods known in the art.

It will be appreciated as described below, that a variety of inventiveconjugates can be synthesized according to the methods described herein.The starting materials and reagents used in preparing these compoundsare either available from commercial suppliers such as Aldrich ChemicalCompany (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis,Mo.), or are prepared by methods well known to a person of ordinaryskill in the art following procedures described in such references asFieser and Fieser 1991, “Reagents for Organic Synthesis”, vols 1-17,John Wiley and Sons, New York, N.Y., 1991; Rodd 1989 “Chemistry ofCarbon Compounds”, vols. 1-5 and supps, Elsevier Science Publishers,1989; “Organic Reactions”, vols 140, John Wiley and Sons, New York,N.Y., 1991; March 2001, “Advanced Organic Chemistry”, 5th ed. John Wileyand Sons, New York, N.Y.; Larock 1990, “Comprehensive OrganicTransformations: A Guide to Functional Group Preparations”, 2^(nd) ed.VCH Publishers; and other references more specifically drawn to polymerchemistry. The methods described below are merely illustrative of somemethods by which the conjugates of this invention can be synthesized,and various modifications to these methods can be made and will besuggested to a person of ordinary skill in the art having regard to thisdisclosure.

The starting materials, intermediates, and conjugates of this inventionmay be isolated and purified using conventional techniques, includingfiltration, distillation, crystallization, chromatography, and the like.They may be characterized using conventional methods, including physicalconstants and spectral data

Materials

Sodium borohydride, sodium cyanoborohydride, sodium metaperiodate,1-[3-(dimethylamino)propyl-3-ethylcarbodiimide hydrochloride (EDC),diethylenetriaminepentacetic acid (DTPA), 4-dimethylaminopyridine (DMAP)and succinic anhydride were from Aldrich, St Louis, Mo. InCl₃ [In-111]was from Perkin Elmer Life Sciences (Boston, Mass.). Anhydrous pyridine,ethyl alcohol, and other solvents were obtained from Sigma-Aldrich andused without further purification.

Camptothecin was obtained from Hande Tech development Co. (Houston,Tex.). Dextran B-512 (Mn 73,000 Da, 188,000 Da) and N-BOC-glycine wereobtained from Sigma Chemical Company (St Louis, Mo.). Succinic anhydride(SA), sodium borohydride, sodium metaperiodate,1-[3-(dimethylamino)propyl-3-ethylcarbodiimide hydrochloride (EDC),diisopropylcarbodiimide (DIPC), 4-dimethylaminopyridine (DMAP),trifluoroacetic acid, hydrochloric acid and sodium hydroxide werepurchased from Aldrich (St Louis, Mo.). Other chemicals, of reagent orhigher grade, were obtained from Acros Organics or Fisher Scientific andused as received. Anhydrous pyridine, methyl alcohol, ethyl alcohol,dimethylformamide, dimethylsulfoxide, methylene chloride, diethyl etherand other solvents were obtained from Sigma-Aldrich and used withoutfurther purification. Deionized water (resistivity>18 MΩ) was used forall synthetic and analytical procedures.

Equipment and Methods

Size exclusion chromatography (SEC) in aqueous media and reversed phase(RP) chromatography were carried out using a Varian-Prostar HPLC systemequipped with a BIO-RAD model 1755 Refractive Index detector andLDC/Milton Roy SpectoMonitor 3000 UV detector. HPSEC Biosil SEC-125 andBiosil SEC400 (BIO-RAD), and low-pressure Superose-6 (Pharmacia) columnswere used for size exclusion chromatography. SEC column calibration wasperformed based on broad molecular weight dextran and protein standards.Unless otherwise stated, elution was performed isocratically with 50 mMphosphate buffered 0.9% NaCl, pH=7.0.

An Altima C18 column (Alltech, 250 mm×4.6 mm, 5 μm bead) was used for RPchromatographic determination of low molecular weight CPT derivativesand degradation products of polymer-CPT conjugates.

Preparative isolation and purification of polymers and polymerconjugates was carried on a G-25 gel SpectraChrom (60 cm×ID 10 cm)column equipped with a Milton-Roy liquid delivery system, MasterFlex CLperistaltic pump, Knauer-2401 RI detector, Foxy JR fraction collectorand Varian-Prostar data acquisition system. Alternatively, a QuixStendflow dialysis system (A/G Technology, Needham, Mass.) equipped with aUFP-10-C-4MA hollow fiber cartridge (cutoff 10 kDa) was used in highvolume procedures. Photon correlation light scattering was carried outusing a Brookhaven ZetaPlus analyzer.

Proton and ¹³C NMR were carried out on Varian Mercury-300, BrukerDPX-300, and Bruker Aspect 3000 NMR spectrometers using solvent peak asreference standard.

A Cary 300Bio UV-visible spectrophotometer equipped with thermostatedmulti-cell Peltier block, and Molecular Devices Co. 96-well Plate Readerwas used for spectroscopic measurements and enzyme kinetics studies.

An Agilent 1100 series LC/MSD system was used for MS characterization ofPHF-CPT hydrolysis products.

Male nu/nu mice, 18-24 g (8-10 week of age) were obtained from CharlesRiver Laboratories, MA.

Human colorectal adenocarcinoma HT-29 cell culture was from ATCC (ATCCHTB-38).

Photoimaging was carried out using Nikon Eclipse TE300 microscope withlong working distance phase contrast optics, epifluorescence imagingsetup, CCD camera, and MacOS based imaging workstation.

Radioactivity measurements were carried out using Wallac Wizard 1480gamma counter (Perkin Elmer). Gamma scintigraphy was performed usingOhio Nuclear gamma camera with medium energy collimator.

PHF-CPT Conjugates

As discussed above, biodegradation of macromolecular therapeutics is animportant but incompletely studied issue, even for most widely usedpolymers. For example, there is a potential risk that extended clinicaluse of conjugates containing non- or slow-biodegradable polymerfragments can lead to long-term cell vacuolization (see, for example,Bendele A. Seely J. Richey C. Sennello G. Shopp G. (1998) Shortcommunication: renal tubular vacuolation in animals treated withpolyethylene-glycol-conjugated proteins. Toxicological Sciences. 42,152-7) and overload, development of lysosomal disease syndrome (see, forexample, Christensen, M., Johansen, P., Hau C., (1978) Storage ofpolyvinylpirrollidone (PVP) in tissue following long-term treatment witha PVP-containing Vasopressin preparation. Acta Med. Scand., 204,295-298), and, at higher doses, to other pathological metabolicalterations (see, for example, Miyasaki K. (1975) Experimental PolymerStorage Disease in Rabbits. Virchows Arch. A. Path. Anat. And Histol.,365, 351-365). Development of essentially completely biodegradablepolymers, preferably degrading with formation of low-toxicity, readilyclearable or metabolizable products, appear to be the predominantpossible radical solution of the problem of long-term intracellulardeposition. A combination of a macromolecular material and across-linking reagent enabling sufficient conjugate stability in thenormal extracellular environment and, on the other hand, acceptable rateof conjugate disintegration upon endocytosis, would be most beneficial.

Hydrophilic essentially fully degradable polyals, e.g.,poly[1-hydroxymethylethylene hydroxymethyl-formal] (PHF), have beendeveloped and reported as acyclic mimetics of polysaccharides (see, forexample, (1) Papisov M I, Garrido L, Poss K, Wright. C, Weissleder R,Brady T J. (1996) A long-circulating polymer with hydrolizable mainchain. 23-rd International Symposium on Controlled Release of BioactiveMaterials, Kyoto, Japan, 1996; Controlled Release Society, Deerfield,Ill.,; 107-108; and (2) Papisov M. I. (1998) Theoretical considerationsof RES-avoiding liposomes. Adv. Drug Delivery Rev., 32, 119-138). Thesematerials, which can be prepared synthetically and by lateral cleavageof some polysaccharides, were shown to be essentially (i)non-bioreactive, (ii) non-toxic and (iii) fully degradable, and, thus,proved to have potential in various pharmaceutical applications (see,for example, (1) Papisov M I, Babich J W, Dotto P, Barzana M, Hillier S,Graham-Coco W, Fischman A J. (1998) Model cooperative (multivalent)vectors for drug targeting. 25th Int. Symp. on Controlled Release ofBioactive Materials, 1998, Las Vegas, Nev., USA; Controlled ReleaseSociety, Deerfield, Ill., 170-171; and (2) Papisov M I. (2001) Acyclicpolyacetals from polysaccharides. (Biopolymers from polysaccharides andagroproteins), ACS Symposium Series 786, pp. 301-314). Polyals containpH-sensitive acetal or ketal groups within the main chain, whichprovides the desired combination of polymer stability in neutral andalkaline media and destabilization in acidic environment.

In certain embodiments, the present invention further expands the scopeof potential applications for hydrophilic polyals, and demonstratessuitability of these materials for preparation of essentially fullydegradable carrier-drug conjugates in dual phase drug release systems.In certain exemplary embodiments, a hydrophilic polyal (PHF) is used toobtain and characterize PHF-CPT conjugates.

Camptothecin¹ (CPT) is a potent antineoplastic agent with topoisomeraseI inhibiting activity. Therapeutic application of unmodified CPT ishindered by very low solubility in aqueous media, high toxicity, andrapid inactivation through lactone ring hydrolysis in vivo. Lactonehydrolysis, which is reversible in acidic media, leads to a watersoluble carboxylate.² The latter is cleared by the kidneys and causeshemorrhagic cystitis, a severe adverse reaction to CPT administration.Acylation of the (O20) lactone ring hydroxyl significantly increases thestability.^(3,4)

Hydrophilization of the CPT molecule results in water soluble forms,e.g. Irinotecan (CPT-11). The latter is the most widely used solubleprodrug, which (as well as other CPT prodrugs) require endoplasmicactivation, mainly in the liver, for conversion into the active form(SN38⁵). Such prodrugs, activated outside cancer tissue, are notfeasible for tumor as well as cancer cell targeting.

Macromolecular and liposomal forms of CPT have shown improved efficacy,as compared to low molecular weight analogs.^(6,7) However, bladdertoxicity was still reported.⁸ The dual phase drug release systemdescribed in this paper was intended to engineer soluble, potentiallytargetable macromolecular preparations with novel pharmacokinetics andreduced toxicity.

The dual phase strategy involves assembling of a hydrophilic conjugatethat releases a lipophilic stabilized CPT prodrug, which, in turn,releases the active drug substance locally (intra- and extracellularly),without the need for prior metabolization by the hepatic microsomal P450complex.

The model release system developed in this work is based on a knownreaction, hydrolytic cyclization of succinamidoesters. The reactionresults in ester bond cleavage and simultaneous succinimide formation atthe amide side. Attempts have been made to employ succinamidoesterlinkers with the amide group at the carrier side,⁹ which does not resultin dual phase drug release. In our system, the succinamidoester isoriented such that the ester is formed at the polymer side, while theopposite carboxyl forms an amide bond with an amine-containing drug ordrug derivative (in this paper, CPT-(O20)-glycinate).

Hydrolysis of the succinamidoester linker leads to drug cleavage fromthe polymer in the form of a cyclic succinimidoglycyl-CPT (Scheme 2).The reaction is base catalyzed, and in aqueous medium goes to completionunder mild conditions. The second stage is in vivo glycyl ester bondhydrolysis, which results in active drug release.

Potential advantages of this dual phase drug release system, as appliedto CPT, include: (1) The conjugate is water soluble, and can beadministered intravenously. (2) Unlike other CPT prodrugs, e.g.Irinotecan, the intermediate prodrug is activated “on site” rather thanin the liver, so that local administration and targeting are possible.(3) CPT is released in a lipophilic, lactone-stabilized form, whichensures prodrug deposition in tissues and low rates of redistributionand carboxylate transfer to urine.

In this paper, a model fully biodegradable macromolecular CPT conjugatewith dual phase release from an unsubstituted succinamidoester linkerwas synthesized and characterized in vitro. Initial results of ongoingin vivo characterization studies are also presented.

The conjugate was assembled using poly(1-hydroxymethylethylenehydroxy-methyl formal) (PHF) as a backbone. PHF is a highly hydrophilic,biodegradable “stealth” polymer developed in our laboratory.^(10,11)Biodegradability of PHF reduces the potential risks associated withadministration of large doses of non-degradable polymers, making themodel PHF conjugate feasible for clinical development.

EXAMPLE 1 PHF

PHF is a semi-synthetic acyclic polyacetal prepared by exhaustivelateral cleavage of Dextran B-512. Complete periodate cleavage of the(1->6)-polyglycoside sequence of Dextan B-512 results inpoly(1-carbonylethylene carbonyl formal) (PCF). Borohydride reduction ofthe pendant aldehyde groups of PCF gives poly(1-hydroxymethylethylenehydroxy-methyl formal) (PHF), a copolymer (copolyacetal) of glycerol andglycol aldehyde (See structure below). Incomplete cleavage at theoxidation stage results in the presence of vicinal glycol groups inplace of some of the methylol groups, which is in some instancesdesirable. An even lower degree of cleavage results in the presence ofboth glycol groups and some intact carbohydrate rings in the polymerchain.

Properties of PHF include the following:

PHF is a highly hydrophilic, water soluble polymer, stable inphysiological conditions, but undergoing proton-catalyzed hydrolysis atlysosomal pH.

The polymer showed no toxicity in mice at doses up to 4 g/kg IV and IP(higher doses not studied). Upon IV administration, low molecular weightPHF (<50 kDa) is almost completely cleared by kidneys with nosignificant accumulation in any tissues.

High molecular weight PHF and derivatives (PHF modified macromoleculesand model drug carriers) that are not cleared by kidneys circulate withhalf-lives up to 10-25 hours (rodents), with a nearly uniform finaldistribution (accumulation per g tissue in RES only twice higher than inother organs). The latter suggests lack of recognition by phagocytes,other cells and recognition proteins (“stealth” properties²⁷)

PHF was prepared, at multi-gram scale, in a variety of molecularweights. The chemical structure of PHF enables a wide variety ofmodifications and derivatizations, via pendant OH groups as well as viaat least one terminal vicinal glycol group.²⁸ Several PHF derivativeswere synthesized and characterized as model biomedical preparations(protein and small molecule conjugates, gels, long-circulating drugcarriers, etc.).^(28,29,30,31,32,33,34)

Due to the “stealth” properties, biodegradability profile, andtechnological flexibility, PHF is a highly promising material forseveral pharmaceutical and bioengineering applications. In particular,the biodegradability and multifunctionality of PHF eliminate severallimitations on the size and structure of small molecule conjugates,enabling, for example, high dose administration of high molecular weightconjugates (>50 kDa) without the risk of long term polymer depositionsin cells.

Several clinically relevant model preparations of PHF developed in ourlaboratory at MGH were evaluated in collaborative studies with thepharmaceutical industry. Various aspects of polyacetal technology wereco-developed with, or licensed by MGH to Novartis, Amgen, andNanopharma.

Additional guidance for the preparation of PHF polymeric material can befound, inter alia, in PCT/US03/22584.

The polymer was prepared using an accelerated modification of apreviously described technique¹² allowing the formation of PHF with 5%2,3-dihydroxyethylformal units originating from the C2-C3 of dextran.Dextran B-512, 73 kDa preparation (15.15 g, 93.4 mmol byglycopyranoside), was dissolved in 300 ml of deionized water at 0-5° C.and treated with 47.95 g (224.2 mmol) of sodium metaperiodate in a lightprotected reactor for 3 hours. The crystalline sodium iodate was removedfrom the reaction mixture by filtration (1μ glass filter). The pH of thefiltrate was adjusted to 8.0 with 5N NaOH and the resultant solution wasimmediately treated with sodium borohydride (7.07 g, 187 mmol, dissolvedin 70 ml of deionized water) for 2 hours. The pH was then adjusted to6.5 with 1 N HCl. The product was desalted on Sephadex G25 andlyophilized; yield: 80%. The results of SEC analysis were Mn=60 kDa andpolydispersity index (Mw/Mn) of 2.0. Proton NMR spectrum in DMF-d₆:D₂O(95:5 v/v) was found to be in agreement with the expected PHF structure(C1-H at δ 4.62 t, J=5.2 Hz) with ca 5% vicinal diol pendant groupsoriginating from C2-C3(δ 4.49 d, J=5.2 Hz).

EXAMPLE 2 CPT-20-(O)-glycinate trifluoroacetate salt (CPT-Gly.TFA)

CPT-Gly.TFA, was prepared in two steps according to the procedurereported by Greenwald^(12,14) and modified by Minko^(1e). Briefly, CPTwas treated with BOC-glycine and DIPC in methylenechloride in thepresence of DMAP. The N-BOC group was removed with trifluoroacetic acid,and the resultant CPT-Gly.TFA was crystallized from diethyl ether.Purity: >97% (HPLC, NMR).

EXAMPLE 3 PHF-Succinate (PHF-SA)

PHF (10.00 g, 75.6 mmol), succinic anhydride (0.76 g, 7.6 mmol) and DMAP(1.2 mg, 0.01 mmol) were dissolved in 5 ml of anhydrous pyridine. After18 hours of agitation at 40° C., pyridine was removed in vacuum, theresidue was suspended in deionized water, and the pH was adjusted to 7.0with 1 N NaOH. The succinylated PHF was desalted on Sephadex G-25 andlyophilized with 86% yield. The succinic acid content, as determined bypotentiometric titration, was 10.3% (mol/monomer). The ¹H NMR spectrumof the polymer (D₂O) contained signals characteristic for methyleneprotons of succinic acid ester at δ 2.66 and δ 2.57 (broad triplets) inaddition to methylene and methine (δ 3.3-3.8), and acetal (δ4.4-4.7)protons of the PHF backbone.

EXAMPLE 4 Camptothecin-PHF Conjugate (PHF-CPT)

Conjugation of CPT-Gly.TFA with PHF-SA was conducted via (i) EDCmediated amidation of polymer-succinate with CPT-20-O-glycinatetrifluoroacetate salt in aqueous medium, or (ii) DIPC mediated couplingin non-aqueous conditions (DMF). The first approach (described below)was found to be more efficient, based on higher reaction rate, cleanerproduct and simplicity of purification.

Prior to the preparative synthesis, conjugates with various CPT contents(ca. 5% to 15% w/w) were prepared on a lower scale to test solubility inaqueous media, which showed that conjugates with CPT content up to 10%w/w were readily soluble.

Preparative synthesis. PHF-SA (15.0 g, 10.7 mmol SA) was dissolved in150 ml of deionized water and mixed with 30 ml of DMF, cooled to −2° C.,and combined with CPT-Gly.TFA solution (2.0 g/3.85 mmol in 20 ml of 3:1acetonitrile/water mixture). Under intense agitation, EDC (2.0 g) wasadded to the reaction mixture. The pH was adjusted to 5.9-6.0. After 30minutes of agitation, the temperature of the reaction mixture wasbrought to ambient, and agitation was continued for another 3 hours. TheCPT conversion at this point was 93%, based on RP HPLC (UV at 360 nm).The pH was adjusted to 5.5 to prevent CPT release from the conjugate,and the reaction mixture was stored overnight at 8° C. The mixture wasthen diluted with DMF and water to 600 ml (DMF content 10% v/v), and theconjugate was desalted on Sephadex G-25, lyophilized, and stored at −20C.°. The product was obtained as an off-white to pale-yellow solid withCPT content of 7.48% w/w (as determined spectrophotometrically at 360nm). Yield based on CPT: 80%.

Proton NMR spectrum of PHF-CPT (DMSO-d₆/D₂O) contained the signalscharacteristic for the succinic acid modified PHF backbone: δ 3.3-3.8(methylene and methine), δ4.4-4.7 (acetal), δ 2.4-2.6 (—CH₂—,succinate); and signals corresponding to the pendant CPT structures: δ0.95 (t), δ 2.21 (d), δ 5.26(m), δ 5.46(s), δ 7.20(s), δ 7.70(t), δ7.88(t), δ 8.09(d), δ 8.18(d), δ 8.45(s).

The reaction mixture and lyophilized product compositions are shown inTable 1. TABLE 1 PHF-CPT conjugate composition (by CPT, mol %). CPTReaction Isolated # derivatives mixture product 1 PHF-CPT 92.8 96.15 2CPT-Glycinate 1.9 0.32 3 CPT 2.3 0.34 4 CPT-CA 0.3 0.44 (carboxylate) 5CPT-Gly-SA <0.05 0.53 6 CPT-Gly-SI <0.05 0.66 7 Other low MW 2.7 1.59

The synthesized PHF-CPT was soluble in aqueous media. HPSEC showed Mn of˜65 kDa with essentially no aggregation (photon correlation lightscattering). The viscosities of up to 20% solutions were feasible forinjection through a high gauge needle used in the rodent studies; mostinjections were performed at 6% w/w (η=4.05 cps).

EXAMPLE 5 Camptothecin-20-(N-succinimidoglycinate) (CPT-SI)

CPT-SI is the lipophilic prodrug isolated from the products of PHF-CPThydrolysis (see below). CPT-SI was synthesized as a control compound.

PHF-CPT (500 mg) was dissolved in 10 ml of 0.1M phosphate pH 7.6 andincubated for 24 hours at 37° C. The resultant suspension was diluted to150 ml and extracted with methylene chloride (3×150 ml). Methylenechloride layers were combined, washed with 0.01 N HCl, and dried overmagnesium sulfate. Solvent was removed in vacuum. The light yellowresidue was redissolved in methylene chloride, filtered and dried invacuum to yield 38 mg of a product containing, according to RPHPLC, >93% CPT-SI. Solubility of CPT-SI in water was found to be lowerthan that of unmodified CPT, <1.0 μg/ml, vs. 2.5 μg/ml respectively.

¹H NMR (300 MHz, CDCl₃): δ 1.01(τ, 3H, J=7.4 Hz, C19), δ 2.05-2.32 (m,2H, C18), δ 2.66 (s, 4H, succinimide), δ 4.32-4.51(AB, 2H, 17.2 Hz, C-αGly), δ 5.32 (s, 2H, C-5), δ 5.29-5.65 (AB, 2H, 17.3 Hz, C-17), δ 7.20(s, 1H, C-14), δ 7.60 (t, 1H, J=7.5 Hz, C-11), δ 7.76 (t,1H, J=7.7 Hz),δ 7.86 (d, 1H, J=8.3, C-12), δ 8.20 (d, 1H, J=8.3, C-9), δ 8.32(s, 1H,C-7)

¹³C NMR: 7.23, 28.36, 29.89, 32.04, 39.53, 50.17, 67.31, 77.45, 96.29,120.54, 128.23, 128.33, 128.64, 130.00, 130.80, 131.35, 145.14, 146.70,149.08, 152.46, 157.48, 166.27, 166.78, 175.95.

MS: m/z 488.2 (M+H)

EXAMPLE 6 Camptothecin-20-(N-succinamidoglycinate) (CPT-SA, control)

CPT-Gly.TFA (50 mg, 0.096 mmol) and succinic anhydride (18 mg, 0.190mmol) were dissolved in 2 ml of anhydrous pyridine. After an 18 houragitation at ambient temperature, pyridine was removed in vacuum. Thesolid residue was suspended in deionized water and extracted withmethylene chloride, washed with 0.01N HCl and dried over magnesiumsulfate. Solvent removal in vacuum resulted in a light-yellow solid(41.4 mg, 85% yield) containing >90% CPT-SA (HPLC with 360 nmdetection). LC-MS: m/z 506.2 (M+H). The product was used as HPLCstandard for determination of PHF-CPT hydrolysis product composition.

EXAMPLE 7 Preparation of Succinylated Polyacetal Carriers

Polyacetal carriers modified with a substituted succinyl group wereprepared according to a procedure analogous to that described above forPHF-SA. Briefly, treatment of anhydrous PHF Mn 60 kDa (10.0 g) in 100 mlof dry pyridine with calculated amount of succinic anhydride derivative(see Table 2) and DMAP (anhydride:DMAP=1:0.1 mol ratio) for 18 hours at40° C. afforded quantitative conversion of succinic acid derivative intocorresponding PHF-succinate with degree of PHF structural unitsubstitution of approximately 10% (mol). After pyridine evacuation invacuum PHF-succinates were dissolved in DI water and purified of lowmolecular weight impurities by gel filtration on Sephadex G-25 columnequilibrated with DI water. Final product was recovered from aqueoussolution by lyophilization as foam with average 85-90% yield. Theobtained PHF-succinates are hydrophilic polymers readily soluble inwater and polar organic solvents (pyridine, DMF, DMSO). Polymer yield,composition, and succinate content are reported in Table 2. TABLE 2Composition and properties of succinylated PHF carriers PolymerSuccinate substitution content* Modified Succinic acid (calculated)mol/g Polymer polyacetal derivative % mol polymer yield, % PHF-SASuccinic 10 7.0 × 10 − 4 86 anhydride PHF-MSA Methylsuccinic 10 6.9 × 10− 4 88 anhydride PHF-DMSA 1,1- 10 6.8 × 10 − 4 85 Dimethylsuccinicanhydride PHF-NSA (2-Nonen-1-yl) 15 8.9 × 10 − 4 89 succinic anhydridePHF-DSA (2-Dodecen-1-yl) 15 8.6 × 10 − 4 91 succinic anhydride*Potentiometric titration

EXAMPLE 8 (2-Nonen-1-yl)-succinic Acid-Linked PHF-CPT (PHF-NSA-CPT)

PHF-(2-nonenylsuccinate) (PHF-NSA) (2.5 g, 2.23 mmol NSA) was dissolvedin 50 ml of deionized water and mixed with 20 ml of DMF, cooled down to0° C., and combined with CPT-Gly.TFA solution (454 mg/0.848 mmol) in 15ml of 4:1 acetonitrile/water mixture). Under intense agitation, EDC (500mg) was added to the reaction mixture. The pH was adjusted to 5.9-6.0.After 30 minutes of agitation, the temperature of the reaction mixturewas brought to ambient; agitation continued for another 3 hours. TheCPT-Gly.TFA conversion after 3 hours monitored by HPLC (UV at 360 mm)was >92%. The reaction mixture was then diluted with 1:9 v/v DMF/watermixture to 150 ml, and the pH of the resulting solution was adjusted to5.5. The obtained conjugate was desalted on Sephadex G-25 andlyophilized. The product was obtained as off-white to pale-yellow solidsoluble in water and polar organic solvents (pyridine, DMF, DMSO). CPTconjugate content determined spectrophotometrically at 360 nm was 13.0%w/w. Yield based on CPT: >95%. Residual carboxyl group content in theconjugate was 4.1×10⁻⁴ mol/g.

Proton NMR spectrum of PHF-NSA-CPT (DMSO-d₆/D₂O) contained the signalscharacteristic for noneneylsuccinic acid modified PHF backbone: δ3.3-3.8 (methylene and methine, PHF), δ4.4-4.7 (acetal), δ 2.6-2.7(—CH₂—, succinate), δ 0.96(t) (—CH₃, noneneyl), δ1.25-1.35 (—CH₂—,noneneyl), δ 5.65 and δ5.75. (—CH═, noneneyl); and signals correspondingto the pendant CPT structures: δ 0.95 (t), δ 2.22 (d), δ 5.26(m), δ5.46(s), δ 7.20(s), δ 7.71(t), δ 7.89(t), δ 8.10(d), δ 8.18(d), δ8.45(s).

EXAMPLE 9 Methylsuccinic acid-linked PHF-CPT (PHF-MSA-CPT)

PHF-(methylsuccinate) (PHF-MSA) (2.5 g, 1.72 mmol MSA) was dissolved in50 ml of deionized water and mixed with 20 ml of DMF, chilled down to 0°C., and combined with CPT-Gly.TFA solution (450 mg/0.840 mmol) in 15 mlof 4:1 acetonitrile/water mixture). Under intense agitation, EDC (500mg) was added to the reaction mixture. The pH was adjusted to 5.9-6.0.After 30 minutes of agitation, the temperature of the reaction mixturewas brought to ambient; agitation continued for another 3 hours. TheCPT-Gly.TFA conversion after 3 hours monitored by HPLC (UV at 360 nm)was >90%. The reaction mixture was then diluted with 1:9 v/v DMF/watermixture to 150 ml, and the pH of the resulting solution was adjusted to5.5. The obtained conjugate was desalted on Sephadex G-25 andlyophilized. The product was obtained as off-white to pale-yellow solidsoluble in water, saline and polar organic solvents (DMF, DMSO),intrinsic pH 5.7. CPT conjugate content determinedspectrophotometrically at 360 nm was 7.65% w/w. Yield based on CPT: 71%.Residual carboxyl group content in the conjugate was 3.0×10⁻⁴ mol/g.

Proton NMR spectrum of PHF-NSA-CPT (DMSO-d₆/D₂O) contained signalscharacteristic for methylsuccinic acid modified PHF backbone and pendantCPT structures.

EXAMPLE 10 1,1-Dimethylsuccinic acid-linked PHF-CPT (PHF-MSA-CPT)

PHF-(1,1-dimethylsuccinate) (PF-DMSA) (2.5 g, 1.70 mmol DMSA) wasdissolved in 50 ml of deionized water and mixed with 20 ml of DMF,chilled to 0° C., and combined with CPT-Gly.TFA solution (450 mg/0.840mmol) in 15 ml of 4:1 acetonitrile/water mixture). Under intenseagitation, EDC (500 mg) was added to the reaction mixture. The pH wasadjusted to 5.9-6.0. After 30 minutes of agitation, the temperature ofthe reaction mixture was brought to ambient; agitation continued foranother 3 hours. The CPT-Gly.TFA conversion after 3 hours monitored byHPLC (UV at 360 nm) was >90%. The reaction mixture was then diluted with1:9 v/v DMF/water mixture to 150 ml, and the pH of the resultingsolution was adjusted to 5.5. The obtained conjugate was desalted onSephadex G-25 and lyophilized. The product was obtained as off-white topale-yellow solid soluble in water, saline and polar organic solvents(DMF, DMSO), intrinsic pH 5.7. CPT conjugate content determinedspectrophotometrically at 360 nm was 6.9% w/w. Yield based on CPT ca.65%. Residual carboxyl group content in the conjugate was 2.9×10⁻⁴mol/g.

Proton NMR spectrum of PHF-NSA-CPT (DMSO-d₆/D₂O) contained the signalscharacteristic for dimethylsuccinic acid modified PHF backbone andpendant CPT structures.

EXAMPLE 11 PHF-CPT Hydrolysis

The hydrolytic stability of PHF-CPT conjugate was tested in DI water andisotonic saline at ambient temperature and pH=5.7, in 0.05M phosphatebuffered 0.9% saline (pH 7.4), and in freshly prepared rat plasma at 37°C. PHF-CPT hydrolysis and accumulation of CPT derivatives was monitoredby RP HPLC using a 20-minute 10-70% acetonitrile/water gradient (bothsolvents with 0.1% TFA). Results were reproduced in two independentexperiments.

The second stage hydrolysis of CPT-SI was investigated analogously.

The reaction of cyclization-elimination (Scheme 2) involves folding ofthe succinamidoester into a cyclic intermediate structure, withsubsequent intramolecular nucleophilic attack on the ester carbon. Thus,the reaction should be sensitive to the presence of (1) bulkysubstitutents and (2) substitutents altering the charge density oneither of the carboxylic carbons of the linker. The second phase canalso be affected by the substitutents in the succinimide ring of theprodrug. Therefore, substitution in the succinate linker can be apowerful tool for regulation of the drug release profile. Furthermore,substitution in the succinate linker can open the way to regulation ofprodrug properties (hydrophobicity, transmembrane transfer, affinity tocell receptors, etc.), which can further enhance pharmacokinetics.

Other substituted analogs of PHF-CPT synthesized using methyl-,2,2-dimethyl, and 2-nonen-2-yl succinates as described in Examples 7-10.Using procedures analogous to the described above, CPT release fromthese conjugates was investigated in phosphate buffered saline asdescribed above. The conjugates were also tested for cytotoxicity inHT29 cell culture.

The PHF-CPT solutions (intrinsic pH=5.5-5.7 with physiologicallynegligible buffer capacity) showed no significant decomposition after aweek of storage at 8° C. or 24 hours at ambient temperature. At neutraland slightly basic pH (7.0-7.4) and mild conditions (8-37° C.), theconjugate did undergo slow hydrolysis yielding primarilyCPT-20-O-(N-succinimidoglycinate) (CPT-SI). For example, hydrolysis ofPHF-CPT conjugate (2 mg/ml in 0.05M phosphate buffered 0.9% saline pH7.4 for 24 hours) resulted in the quantitative release of CPT fromPHF-CPT, with CPT-SI lactone (87%), CPT carboxylate (8%) and CPT-SAlactone (5%) being the only detectable products. Notably, CPT releasedfrom the prodrug under these conditions was in the carboxylate but notlactone form, suggesting that the lactone ring, which was stable inCPT-SI and CPT-SA, was hydrolyzed during the second stage of CPTrelease.

A similar trend but slightly different composition of hydrolyticproducts was observed in freshly prepared rat plasma, as shown onFIG. 1. This suggests the presence of additional CPT release mechanisms,possibly mediated by interactions with plasma proteins. Cleavage of CPT(all forms) from PHF-CPT was found to be monoexponential, withhalf-release time of 2.2±0.1 hours.

The half-time of the subsequent hydrolysis of CPT-SI was over 20 hrs,depending on the conditions (the exact pH dependence and enzymesensitivity, if any, are to be determined in ongoing studies).

The three synthesized substituted analogs of PHF-CPT were alsoinvestigated to determine the first phase release rates. As expected,the bulky nonenyl group (which sterically hinders linker folding, whichis necessary for hydrolytic cyclization) decreased the release rate,while methyl groups, which stabilize cyclic structures, increased it(Table 3; each result based on two independent experiments, n=4-6 datapoints each; for all numbers SD<10% of the mean, p<0.05). TABLE 3Comparative release rates of modified CPT-PHF conjugates in PBS at pH7.4/37° C. CPT half- release time, Compound Linker hours PHF-CPTGly-succinate 2.1 PHF-MSA-CPT Gly-(methyl succinate) 1.4 PHF-DMSA-CPTGly-(2,2-dimethyl succinate) 0.6 PHF-NSA-CPT Gly-(2-nonen-2-ylsuccinate) 16.0

EXAMPLE 12 PHF-SAG-Taxol Conjugate

The water soluble Taxol conjugate with PHF utilizing dual-phase releasesuccinamidoglycine linkage, PHF-succinamidoglycine-Taxol conjugate(PHF-SAG-Taxol), has been prepared from Taxol-2′(O)-glycinate andsuccinilated PHF.

Taxol-2′(O)-glycine-NH₂ was obtained in two steps via acylation of Taxolwith HO-Gly-(Z) (DIPC, DMAP, CH₂Cl₂) followed by amino groupdeprotection (H₂, Pd/C, MeOH) with overall 60% yield [Z=Cbz].Taxol-2′(O)-Gly-NH₂ was conjugated to PHF-succinate (10% mol. succinicacid, MW 65 kDa) via EDC mediated coupling in 50% aqueous DMF.PHF-SAG-Taxol conjugate synthesis was carried out on a 2-gram scale, atambient temperature, pH 5.5-6.0. A quantitative (>98%) Taxol-glycinateconversion to PHF-SAG-Taxol was detected within 3 hours. ThePHF-SAG-Taxol was purified of low molecular weight impurities by gelfiltration on G25 Sefadex column equilibrated with DI water, andrecovered by lyophilyzation. Following the above procedure, conjugateswith Taxol load ranging from 6% to 13% (wt.) were prepared. All productswere readily soluble in deionized water and saline. Stability ofPHF-Taxol conjugates in aqueous media was monitored by HPLC. Aqueoussolutions of PHF-SAG-Taxol were stable at ambient conditions in pH rangefrom 4.5 to 5.5. At physiological conditions (PBS, pH 7.4, 37° C.) thedrug was released from the conjugate with a half-life of 1.5±0.2 hours,resulting in a mixture of Taxol-2′(O)-(succinimidoglycine) and Taxol ata ratio of 1.5:1.0. Under these conditions,Taxol-2′(O)-(succinimidoglycine) ester hydrolyzed to Taxol withhalf-life of approximately 3 hours. Antitumor activity of PHF-SAG-Taxolpreparation with Taxol content of 13% was tested in vitro with HT-29human colorectal carcinoma cells. Both PHF-Taxol conjugate andunmodified Taxol formulations have shown statistically identical cellgrowth inhibitory efficacy (ED50 15 nM).

EXAMPLE 13 Glycyl-Illudin

Illudin M, 50 mg (0.2 mmol), was dissolved in 2 mL of anhydrous THF andcooled to 0° C. Then, 65 mg (0.22 mmol) of Fmoc-glycine, 30 mg (0.22mmol) of diisopropyl carbodiimide, and 1 mg of 4-(dimethylamino)pyridine(DMAP) were added. The reaction mixture was stirred at 0° C. for 2hours, then overnight at ambient temperature. The resultantFmoc-glycyl-illudin was purified by column chromatography (silica,chloroform with 1% ethanol) and dried in vacuum. Yield: 73 mg (70%).

Fmoc-glycyl-illudin (30 mg) was dissolved in 5 mL of 20% piperidine inDMF. The solution was stirred at ambient temperature for 3 hours. Thesolvent was removed under vacuum, and the resultant glycyl-illudin waspurified by column chromatography (silica, chloroform with 3% ethanoland 1% triethylamine). Yield: 10 mg (57%).

EXAMPLE 14 PHF-Illudin M

Anhydrous PHF, Mn 73 kDa (2.0 g), prepared as described in Example 1,was dissolved in 50 ml of dry pyridine. Then, 0.15 g of succinicanhydride and 18 mg DMAP were added. The reaction mixture was incubatedfor 18 hours at 40° C. The reaction resulted in quantitative acylationof PHF with formation of PHF-succinate that had 10% of its monomer unitssuccinylated. After pyridine removal in vacuum, the PHF-succinate wasdissolved in deionized water and purified by gel filtration on aSephadex G-25 column equilibrated with deionized water. The finalproduct was recovered from aqueous solution by lyophilization. Yield:nearly 100%.

PHF-succinate (100 mg) was dissolved in 2 ml of deionized water andmixed with 0.5 ml of DMF. Glycyl-Illudin, 10 mg, was dissolved in 0.5 mLof acetonitrile. The solutions were cooled down to 0° C. and mixed.Under intense agitation, EDC(1-ethyl-3-(3-diethylaminopropyl)carbodiimide; 20 mg) was added to thereaction mixture. The pH was adjusted to 5.9-6.0. After 30 minutes, thetemperature of the reaction mixture was brought to ambient; agitationcontinued for another 3 hours. Glycyl-illudin association withPHF-succinate was monitored by size exclusion HPLC (detection: UV at 318nm). Upon completion of the reaction, 15 ml of deionized water wereadded. The pH was adjusted to 5.5, and the reaction mixture wasimmediately desalted on Sephadex G-25. The product, PHF-Illudin, waslyophilized. Yield: nearly 100%. Polymer properties are reported inTable 4. TABLE 4 Properties of PHF-Illudin M conjugates Sample % drugload¹ Molecular Reasonable number By weight weight solubility² 1 1.5%  78,000 4 mg/mL 2 3% 78,000 7.5 mg/mL 3 4% 78,000 10 mg/mL¹Drug load was determined by UV spectrometer at 318 nm.²The reasonable solubility was determined by dissolved 250 mg conjugatesin 1 mL of water. The viscosity of resulting solution was not too highto do IV injection.

EXAMPLE 15 Labeling

A dual labeled conjugate (³H labeled CPT and ¹¹¹In labeled backbone) wasused for parallel independent monitoring of the conjugate components.

A [³H] labeled PHF-CPT conjugate with 0.210 mCi/g activity and 7.0% w/wCPT content was prepared using [5-³H(N)]-camptothecin (MoravekBiochemicals, Inc.) as described for PHF-CPT. The polymer backbone ofthe conjugate was modified with DTPA and labeled with ¹¹¹In bytranschelation from indium citrate at pH 5.5. Modification of PHF-CPTwith DTPA was carried out in two steps. (1) Vicinal diols present in PHFstructure (see Example 1) were oxidized with sodium metaperiodate atdiol:periodate ratio 1:1, pH 5.7, for 2 hours at ambient temperature.The resultant pendant aldehyde groups were nonreductively aminated withDTPA amide of 1-amino-2-hydroxy-3-(aminooxy)-propan. The latter“aminooxy-DTPA”, which forms oxime bonds with aldehydes under mildconditions, was prepared in our laboratory (synthesis to be describedelsewhere). In our opinion oximes, being significantly more stable underphysiological conditions than hydrazides¹⁵ and generally less toxic, aremore suitable for carbonyl modification in modular conjugates.

Radiochemical purities of all labeled derivatives were >98% (HPLC).

EXAMPLE 16 Biokinetics

Biokinetics and biodistributions of PHF-CPT conjugates were studied innormal rats and in nude mice with HT29 and A2780 xenografts usingconjugates containing double-labeled labeled CPT conjugates. All animalstudies were conducted in accordance with institutionally approvedprotocols.

Male nude/nu mice, average weight 28-32 g (Charles River Labs, Boston,Mass.), bearing 150-200 μl tumor xenografts (n=6 per group) wereinjected iv with the dual-labeled labeled PHF-CPT in 0.9% saline at 20mg/kg based on CPT. The injected activities were 1.25 μCi/animal for ³Hand 5 μCi/animal for ¹¹¹In.

Adult outbred 240 g male rats (Charles River Laboratories, Boston,Mass.), n=6 per group, were injected iv with 800 μl of labeled PHF-CPTin 0.9% saline at 20 mg/kg by CPT. The injected activity per animal was1.25 μCi and 24 μCi for ³H and ¹¹¹In respectively.

Blood samples were taken at 5, 15, 30 minutes and 1, 2, 4, 8 and 24hours time points. At 24 hours, the animals were euthanized; tumors andsamples of major organs were harvested for counting. The total ³H and¹¹¹In activities in tissues were measured by scintillation (beta) andgamma counting respectively, and expressed as % injected dose/g tissueto characterize the distributions of ³H-CPT (total of all forms) and¹¹¹In-PHF.

The carrier polymer half-life in rat was found to be 14.2±1.7 hours,while the drug substance half-life was 2.1±0.2 hours, which correspondswell to the determined in vitro first phase release rate (FIG. 5).

Both ³H-CPT and ¹¹¹In-PHF showed substantial accumulation in the tumortissue. At 24 hours, CPT uptake in the tumor was 2.22% and 2.52% dose/gfor A2780 and HT29, respectively, which is ca. 75-fold higher than forCPT (p<0.05) and very similar to PEG-CPT.¹⁴ Mean tumor to muscle ratioswere 2.4 and 1.5, respectively (p<0.2 for the difference between twodifferent xenografts).

Accumulation in other tissues (FIG. 6) was also similar to that ofPEG-CPT. However, 2-3-fold higher drug levels were detected in thereticuloendothelial system (RES) tissues. Although it is not bound bytheory, the latter could be either due to higher RES uptake of theCPT-SI then PEG-CPT, or due to a blood volume dependentpharmacokinetics.

Photoimaging (fluorescence microscopy) of CPT fluorescence in unstainedunfixed tumor tissue 24 hours post administration showed relativelyhomogenous CPT distribution with elevated drug accumulation in someareas adjacent to vascular beds (FIG. 7). Diffuse intracellulardistribution of CPT fluorescence indicated predominantly cytoplasmic(non-vesicular) drug localization.

EXAMPLE 17 Antiproliferative Activity

Cytotoxicity of CPT derivatives was investigated in HT29 cell culture.Cells were grown in McCoy's 5a medium with 1.5 mM L-glutaminesupplemented with 10% FBS. The (exponentially growing) cells were seededin 24-well culture plates (˜10000 cells/well), cultured for 24 hours,and then treated with test compounds at various dilutions. Growthinhibition was assessed 72 hours post treatment (MTT assay). The ID50 ofPHF-CPT in HT-29 cell culture was found to be 172 nM, which is 10-foldhigher than CPT ID50 (17 nM), and 5-fold higher than CPT-SI ID50 (34nM).

EXAMPLE 18 In Vivo Antineoplastic Activity and Toxicity

The toxicity of CPT was evaluated in normal outbred mice, as well as inxenograft bearing nude athymic animals in the course of antineoplasticactivity studies.

The antineoplastic activity of PHF-CPT was evaluated with a HT-29xenograft model in athymic mice in accordance with institutionallyapproved protocols. Camptothecin and CPT-SI (the first phase releaseproduct) were used as controls.

The study was carried out using approximately equitoxic doses of CPT andPHF-CPT. Cells were injected subcutaneously into the left flank, 10⁶cells per animal in 50 μl. When tumor volume reached 100-150 mm³, micewere randomly divided into four experimental groups: PHF-CPT,camptothecin, CPT-SI, and untreated control (n=3 each). Animals of thefirst three groups received the respective experimental substance viathe tail vein in five doses every three days (5×q3D). Each injectioncontained 22.5 mg CPT eqv/kg of CPT and CPT-SI, and 45 mg CPT eqv/kg forPHF-CPT. All formulations were prepared immediately prior toadministration. PHF-CPT was administered as a solution in 0.9% saline.CPT and CPT-SI were administered as dispersions in Tween 80/water (9/1v/v). Animal weight, tumor size, animal appearance, behavior, andsurvival rate were monitored for four weeks after administration. Weightloss over 20% and tumor growth over 1500 mm³ were counted as lethalities(animals were euthanized).

EXAMPLE 19 In Vivo Activity of PHF-CPT in LS174t Xenograft Model

PHF-CPT was administered IV, 160 nm/kg by CPT, q7dx3 to nude mice withgrowing LS174t tumor xenografts. Irinotecan (a soluble low molecularweight CPT derivative, used as control) was administered IP, also at 160nm/kg, following the same schedule. The results (FIGS. 3 and 4)demonstrated that, at the same doses (by drug substance), PHF-CPTsuppressed tumor growth more potently than Irinotecan (FIG. 3). Thegroup of animals treated with PHF-CPT had better survival rate than thegroup treated with Irinotecan (FIG. 4).

The maximum tolerated dose (MTD) of PHF-CPT was found to be >24 mg/kg,which is at least two-fold higher than for the low molecular weight CPTand Irinotecan (9-10 mg/kg for analogous schedules).

EXAMPLE 20 PHF-CPT Antineoplastic Toxicity

Antineoplastic toxicity of PHF-CPT was tested in the HT29 model (tumorsize 100-150 μL), using CPT and CPT-SI as controls. The latter wereadministered as Cremofor® emulsions. PHF-CPT administered at 45 mg/kg byCPT (5×q3d.) was found to be both more effective and less toxic thanunmodified CPT at 22.5 mg/kg (same schedule). The intermediate releaseproduct, CPT-SI, was found to have no significant effect on tumordynamics, as determined by the time of tumor growth from 0.10-0.15 cm³to 1.5 cm³ (27 days vs. 24 for untreated control and 40 days for CPT at22.5 mg/kg 5×q4d).

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1. A conjugate comprising a carrier substituted with one or moreoccurrences of a moiety having the structure:

wherein each occurrence of M is independently a modifier having amolecular weight≦10 kDa;

denotes direct of indirect attachment of M to linker L^(M); and eachoccurrence of L^(M) is independently an optionally substitutedsuccinamide-containing linker, whereby the modifier M is directly orindirectly attached to the succinamide linker through an amide bond, andthe carrier is linked directly or indirectly to each occurrence of thesuccinamide linker through an ester bond.
 2. The conjugate of claim 1,wherein each occurrence of L^(M) independently comprises a moiety havingthe structure:

wherein

denotes the site of attachment to the modifier M;

denotes the site of attachment to the carrier; q is an integer from 0-4;R¹ is hydrogen, —C(═O)R^(1A), C(═O)OR^(1A), —SR^(1A), SO₂R^(1A) or analiphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl,aromatic, heteroaromatic moiety, wherein each occurrence of R^(1A) isindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aromatic, heteroaromatic, aryl orheteroaryl; and each occurrence of R² is independently hydrogen,halogen, —CN, NO₂, an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl, heteroaryl, aromatic, heteroaromatic moiety, or—GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—,—C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—, —OC(═O)O—,—OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—, —C(═S)—,—C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) is independently hydrogen,halogen, or an optionally substituted aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aromatic, heteroaromatic, aryl or heteroarylmoiety.
 3. The conjugate of claim 2, wherein R¹ is hydrogen or alkyl,alkenyl, —C(═O)R^(1A), —C(═O)OR^(1A), —SR^(1A), SO₂R^(1A); wherein eachoccurrence of R^(1A) is independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aryl or heteroaryl.
 4. The conjugateof claim 3, wherein R¹ is hydrogen.
 5. The conjugate of claim 2, whereineach occurrence of R² is hydrogen.
 6. The conjugate of claim 1, whereinthe carrier is a macromolecule, soluble polymer, nanoparticle, gel,liposome, micelle, suture or implant.
 7. The conjugate of claim 6,wherein the carrier is a polyal.
 8. The conjugate of claim 7, whereinthe polyal is a biodegradable biocompatible polyacetal wherein at leasta subset of the polyacetal repeat structural units have the followingchemical structure:

wherein for each occurrence of the n bracketed structure, one of R¹ andR² is hydrogen, and the other is a biocompatible group and includes acarbon atom covalently attached to C¹; R^(x) includes a carbon atomcovalently attached to C²; n is an integer; each occurrence of R³, R⁴,R⁵ and R⁶ is a biocompatible group and is independently hydrogen or anorganic moiety; and for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a functional groupsuitable for coupling with a succinamide through an ester bond.
 9. Theconjugate of claim 7, wherein the polyal is a biodegradablebiocompatible polyketal wherein at least a subset of the polyketalrepeat structural units have the following chemical structure:

wherein each occurrence of R¹ and R² is a biocompatible group andincludes a carbon atom covalently attached to C¹; R^(x) includes acarbon atom covalently attached to C²; n is an integer; each occurrenceof R³, R⁴, R⁵ and R⁶ is a biocompatible group and is independentlyhydrogen or an organic moiety; and for each occurrence of the bracketedstructure n, at least one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises afunctional group suitable for coupling with a succinamide through anester bond.
 10. The conjugate of claim 7, wherein the carrier has thestructure:


11. The conjugate of claim 1, wherein the modifier is a biomolecule,small molecule, organic or inorganic molecule, therapeutic agent,microparticle, pharmaceutically useful group or entity, macromolecule,diagnostic label, chelating agent, intercalator, hydrophilic moiety,dispersant, charge modifying agent, viscosity modifying agent,surfactant, coagulation agent or flocculant.
 12. The conjugate of claim11, wherein the modifier is a chemotherapeutic moiety.
 13. The conjugateof claim 11, wherein the modifier is camptothecin (CPT) or Taxol. 14.The conjugate of claim 11, wherein the modifier is chemically modifiedso that it comprises a functional group suitable for covalent bindingwith an optionally substituted succinic acid through formation of anamide bond; said succinic acid being conjugated to the carrier throughan ester bond.
 15. The conjugate of claim 14 having the structure:

wherein the carrier is a polyal; each occurrence of M is independently amodifier; p is an integer from 1-12; p is an integer from 1-4; t is aninteger designating the number of modifier moieties conjugated to thecarrier; R¹ is hydrogen, —C(═O)R^(1A), —C(═O)OR^(1A), —SR^(1A),SO₂R^(1A) or an aliphatic, alicyclic, heteroaliphatic, heterocyclic,aryl, heteroaryl, aromatic, heteroaromatic moiety, wherein eachoccurrence of R^(1A) is independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl,heteroaliphatic, heteroalicyclic, aromatic, heteroaromatic, aryl orheteroaryl; and each occurrence of R and R² is independently hydrogen,halogen, —CN, NO₂, an aliphatic, alicyclic, heteroaliphatic,heterocyclic, aryl, heteroaryl, aromatic, heteroaromatic moiety, or—GR^(G1) wherein G is —O—, —S—, —NR^(G2)—, —C(═O)—, —S(═O)—, —SO₂—,—C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—, —NR^(G2)C(═O)—, —OC(═O)O—,—OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—, —NR^(G2)C(═O)NR^(G2)—, —C(═S)—,—C(═S)S—, —SC(═S)—, —SC(═S)S—, —C(═NR^(G2))—, —C(═NR^(G2))O—,—C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—, —NR^(G2)C(═NR^(G3))—,—NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or —SO₂NR^(G2)—, wherein eachoccurrence of R^(G1), R^(G2) and R^(G3) is independently hydrogen,halogen, or an optionally substituted aliphatic, heteroaliphatic,alicyclic, heteroalicyclic, aromatic, heteroaromatic, aryl or heteroarylmoiety.
 16. The conjugate of claim 15, wherein p is
 1. 17. The conjugateof claim 15, wherein p is 1 and R is hydrogen.
 18. The conjugate ofclaim 15, wherein each occurrence of M is CPT or Taxol.
 19. Theconjugate of claim 15, wherein p is 1, R and R¹ are each hydrogen, andeach occurrence of M is CPT or Taxol.
 20. A method for preparing aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

wherein each occurrence of M is independently a modifier;

denotes direct of indirect attachment of M to linker L^(M); and eachoccurrence of L^(M) is independently an optionally substitutedsuccinamide-containing linker, whereby the modifier M is directly orindirectly attached to the succinamide linker through an amide bond, andthe carrier is linked directly or indirectly to each occurrence of thesuccinamide linker through an ester bond; said method comprising stepsof: providing a carrier; providing one or more modifiers; reacting thecarrier with an optionally substituted succinic anhydride having thestructure:

wherein q is an integer from 0-4; and each occurrence of R² isindependently hydrogen, halogen, —CN, NO₂, an aliphatic, alicyclic,heteroaliphatic, heterocyclic, aryl, heteroaryl, aromatic,heteroaromatic moiety, or —GR^(G1) wherein G is —O—, —S—, —NR^(G2)—,—C(═O)—, —S(═O)—, —SO₂—, —C(═O)O—, —C(═O)NR^(G2)—, —OC(═O)—,—NR^(G2)C(═O)—, —OC(═O)O—, —OC(═O)NR^(G2)—, —NR^(G2)C(═O)O—,—NR^(G2)C(═O)NR^(G2)—, —C(═S)—, —C(═S)S—, —SC(═S)—, —SC(═S)S—,—C(═NR^(G2))—, —C(═NR^(G2))O—, —C(═NR^(G2))NR^(G3)—, —OC(═NR^(G2))—,—NR^(G2)C(═NR^(G3))—, —NR^(G2)SO₂—, —NR^(G2)SO₂NR^(G3)—, or—SO₂NR^(G2)—, wherein each occurrence of R^(G1), R^(G2) and R^(G3) isindependently hydrogen, halogen, or an optionally substituted aliphatic,heteroaliphatic, alicyclic, heteroalicyclic, aromatic, heteroaromatic,aryl or heteroaryl moiety; under suitable conditions to form asuccinylated carrier having the structure:

or salt thereof; wherein s denotes the number of succinyl moieties onthe carrier; and reacting the succinylated carrier with one or moremodifier moieties (M), whereby at least one modifier moiety forms anamide bond, either directly or indirectly through a secondary linker,with a succinyl moiety present on the carrier; thereby generating theconjugate having the structure:

wherein R² and q are as defined above;

denotes direct of indirect attachment of M to the succinamide linker;and t is an integer designating the number of modifier moietiesconjugated to the carrier such that t≦s.
 21. The method of claim 20,wherein each occurrence of R² is independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl, heteroaryl,—C(═O)R^(2A) or —ZR^(2A), wherein Z is —O—, —S—, —NR^(2B), wherein eachoccurrence of R^(2A) and R^(2B) is independently hydrogen, or an alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, heteroaliphatic, heteroalicyclic, aryl or heteroarylmoiety.
 22. The method of claim 21, wherein each occurrence of R² isindependently hydrogen or alkyl.
 23. The method of claim 20, wherein inthe step of coupling the succinylated carrier, a subset of thesuccinamic acid sites on the carrier remains unreacted.
 24. The methodof claim 20, wherein the carrier is a biodegradable biocompatiblepolyacetal wherein at least a subset of the polyacetal repeat structuralunits have the following chemical structure:

wherein for each occurrence of the n bracketed structure, one of R¹ andR² is hydrogen, and the other is a biocompatible group and includes acarbon atom covalently attached to C¹; R^(x) includes a carbon atomcovalently attached to C²; n is an integer; each occurrence of R³, R⁴,R⁵ and R⁶ is a biocompatible group and is independently hydrogen or anorganic moiety; and for each occurrence of the bracketed structure n, atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises a functional groupsuitable for coupling with a succinamide through an ester bond.
 25. Themethod of claim 20, wherein the carrier is a biodegradable biocompatiblepolyketal wherein at least a subset of the polyketal repeat structuralunits have the following chemical structure:

wherein each occurrence of R¹ and R² is a biocompatible group andincludes a carbon atom covalently attached to C¹; R^(x) includes acarbon atom covalently attached to C²; n is an integer; each occurrenceof R³, R⁴, R⁵ and R⁶ is a biocompatible group and is independentlyhydrogen or an organic moiety; and for each occurrence of the bracketedstructure n, at least one of R¹, R², R³, R⁴, R⁵ and R⁶ comprises afunctional group suitable for coupling with a succinamide through anester bond.
 26. The method of claim 24 or 25, wherein the functionalgroup is a hydroxyl moiety.
 27. The method of claim 20, wherein thecarrier is PHF.
 28. The method of claim 20, wherein at least oneoccurrence of M comprises CPT or Taxol.
 29. The method of claim 20,wherein each occurrence of M comprises CPT or Taxol.
 30. The method ofclaim 28 or 29, wherein CPT or Taxol is indirectly attached to thesuccinamide linker through a glycine moiety.
 31. A compositioncomprising the conjugate of claim 1 and a pharmaceutically suitablecarrier or diluent.
 32. A composition comprising a conjugate of claim 1,wherein at least one occurrence of M comprises CPT or Taxol, wherein theconjugate is associated with an efficient amount of a therapeutic agent;wherein the therapeutic agent is incorporated into an released from saidconjugate matrix by degradation of the conjugate matrix or diffusion ofthe agent out of the matrix over a period of time.
 33. A compositioncomprising a conjugate of claim 1, wherein at least one occurrence of Mcomprises CPT or Taxol.
 34. A composition comprising a conjugate ofclaim 1, wherein each occurrence of M comprises CPT or Taxol.
 35. Thecomposition of claim 33 or 34 wherein CPT or Taxol is indirectlyattached to the succinamide linker via a glycine moiety.
 36. Thecomposition of claim 32, wherein the composition comprises a PHF-CPTconjugate having n occurrences of the bracketed structure n, koccurrences of the bracketed structure k, and m occurrences of thebracketed structure m:

wherein n, k and m are integers between 10-300, 1-20, and 0-300respectively.
 37. The composition of claim 32, wherein the compositioncomprises a PHF-Taxol conjugate having n occurrences of the bracketedstructure n, k occurrences of the bracketed structure k, and moccurrences of the bracketed structure m:

wherein n, k and m are integers between 10-300, 1-20, and 0-300respectively.
 38. A method of treating a disease or disorder, comprisingadministering to a subject in need thereof an efficient amount of aconjugate comprising a carrier substituted with one or more occurrencesof a moiety having the structure:

wherein each occurrence of M is independently a modifier having amolecular weight≦10 kDa;

denotes direct of indirect attachment of M to linker L^(M); eachoccurrence of L^(M) is independently an optionally substitutedsuccinamide-containing linker, whereby the modifier M is directly orindirectly attached to the succinamide linker through an amide bond, andthe carrier is linked directly or indirectly to each occurrence of thesuccinamide linker through an ester bond; wherein said modifier is asuitable therapeutic agent for treatment of the disease or disorder;wherein said modifier is released from the conjugate by a dual phaseprocess.
 39. The method of claim 38 wherein said modifier is locallydelivered by implantation of said conjugate at a desired site ofdelivery.
 40. The method of claim 38 wherein said modifier is selectedfrom the group consisting of: vitamins, anti-AIDS substances,anti-cancer substances, radionuclides, antibiotics, immunosuppressants,anti-viral substances, enzyme inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics, imaging agents.
 41. The methodof claim 38, wherein at least one occurrence of the modifier is CPT orTaxol.
 42. The method of claim 38, wherein each occurrence of themodifier is CPT or Taxol.
 43. The method of claim 38 further comprisingadministering with the conjugate at least one additional biologicallyactive compound selected from the group consisting of vitamins,anti-AIDS substances, anti-cancer substances, radionuclides,antibiotics, immunosuppressants, anti-viral substances, enzymeinhibitors, neurotoxins, opioids, hypnotics, anti-histamines,lubricants, tranquilizers, anti-convulsants, muscle relaxants andanti-Parkinson substances, anti-spasmodics and muscle contractantsincluding channel blockers, miotics and anti-cholinergics, anti-glaucomacompounds, anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, anti-secretory factors, anticoagulants and/or antithromboticagents, local anesthetics, ophthalmics, prostaglandins,anti-depressants, anti-psychotic substances, anti-emetics, imagingagents, and combination thereof.